Marlowe first appears after Nathan Drake and Victor Sullivan defeat her men in a bar room brawl in London, but are cornered outside. After ugly banter between the three, she forcibly takes what she thinks is Sir Francis Drake's ring, though it turns out later to be a fake. As she goes to leave, Drake and Sully try to follow, but are shot and seemingly killed by Charlie Cutter, who is left behind as punishment.
In a flashback to 20 years earlier, it is revealed that Marlowe commissioned Sully in Cartegena to steal the ring, along with the cipher disk it unlocks. However, a young Nathan Drake steals both before they get to it. He is cornered by Marlowe and her men, who take back the cipher disk; however, he escapes with the ring with the help of Sully who takes him under his wing.
Back in the present, Drake, Sully, Chloe Frazer, and Cutter, who is actually an old friend of Sully's, and acting as a double agent, follow Marlowe's trail to her underground lair, where they retrieve the cipher disk, learn that she's looking for Iram of the Pillars (also known as Ubar), a quest previously undertaken by Sir Francis and T.E. Lawrence, and use the disk to search for clues to its location. They find two separate locations: a chateau in France and a citadel in Syria. Nate and Sully investigate the chateau before joining Chloe and Cutter in Syria, where they learn of Marlowe's background. Marlowe's men ambush them inside the citadel, with Marlowe condemning Cutter for betraying her (although he doubts she ever trusted him to begin with). She tries to burn him alive, but he escapes at the cost of breaking his leg. Chloe stays behind to take care of him, leaving Nate and Sully to continue on to their next destination, Yemen.
In Yemen, Nate and Sully, reunited with Nate's estranged wife, Elena Fisher, discover more clues to the location of Ubar. However, Nate is shot with a hallucinogenic dart by Talbot and then taken to Marlowe. Marlowe threatens Nate as to Ubar's whereabouts, but Nate escapes and chases Talbot as he gets word of Sully's location. Rameses, another one of Marlowe's associates, and his gang of pirates capture Nate, and while Nate escapes from them, Marlowe and her men capture Sully and force him to lead them to Ubar. With Elena's help, Nate follows them by stowing away on a cargo plane, but is discovered by Marlowe's men and the resulting shootout leads to the plane crashing. Stranded in the middle of the Rub 'al Khali desert, Nate spends several days wandering around hopelessly, taunted at one point by Marlowe's voice. A group of Arab nomads eventually rescues Nate, who learns from the leader, Salim, that Ubar was doomed millennia ago when King Solomon cast a brass vessel containing evil Djinn spirits into the depths of the city. Nate and the Arabs ambush Marlowe's convoy the next morning and rescue Sully. Despite this, Marlowe and her remaining men (including Talbot) reach Ubar before Nate and Sully do.
In Ubar, Nate, after drinking from a fountain, hallucinates a vision of Talbot killing Sully and him massacring Marlowe's men, who seem to be possessed by the Djinn, in retaliation. Upon regaining his senses, Nate learns that the waters in Ubar are tainted with a powerful hallucinogenic agent, the result of King Solomon's disposal of the brass vessel and the Djinn. Nate and Sully recall Cutter saying that Marlowe's order seeks to gain power by exploiting their enemies through fear, and Nate realizes she can do this with the evil spirits imprisoned in the vessel.
Now more determined than ever to stop Marlowe, Nate and Sully venture further into the city and find her overseeing the retrieval of the vessel from the waters via winch. When they are caught by Talbot, who knocks out Sully, Nate dives into the water and destroys the winch with a flare gun; with two more shots, he also accidentally blows up the central cistern of Ubar, causing the city to start collapsing into a giant sinkhole. Marlowe and Talbot cross paths with Nate and Sully as both sides race to escape the crumbling city, but before anything else can happen, the floor gives way, dropping Nate, Marlowe, and Talbot into a quicksand pit in the room below. While Nate and Talbot are able to reach stable ground, Marlowe, who landed in the middle of the pit, is trapped and sinking fast.
Marlowe calls out to Nate, holds out Sir Francis' ring, urging him to help her and earn the ring as his ancestor did and "prove his greatness"; Nate replies that he has nothing to prove. Despite this, he throws her his belt and tries to pull her to safety, but Marlowe is unable to hold on. Realizing she is doomed, Marlowe resigns herself to her fate as she sinks out of sight, never to be seen again. Talbot, devastated, tries to kill Nate and Sully afterward, only to join his mistress in death. Salim then rescues Nate and Sully as Ubar is finally engulfed by the desert sands.
- WatchMojo.com listed Katherine Marlowe as #6 on their Top 10 "Female Video Game Villains" list.
- She is the only main antagonist in the franchise who is both female and physically fought the protagonist.