Section 31 attempted to recruit the genetically-enhanced chief medical officer of station Deep Space 9, Julian Bashir, in late 2374, after putting him through psychological testing on the holodeck of a ship to which he had been abducted. The commanding agent of that operation was a man known as Luther Sloan, who described Section 31 as an autonomous agency created as part of the original Starfleet Charter. Bashir, appaled at the thought of an agency that is accountable to no one and violates the principles upon which the Federation was founded as a matter of course, rejected Sloan's offer, though Section 31 continued to regard him as an agent. (The use of the term "original" suggests, then, that Thirty-One's origins may go back as far as the Earth Starfleet of Enterprise.) Bashir alerted the command staff of DS9, including his CO, Captain Benjamin Sisko, and those members of the command staff who were officers in the Bajoran Militia, Constable Odo and Major Kira Nerys, of Thirty-One's existence and attempts to recruit him; Odo likened Thirty-One to the United Federation of Planet's equivalent of the Cardassian Union's Obsidian Order, or the Romulan Star Empire's Tal Shiar—the Federation secret police. Sisko and Bashir planned to use any future contacts with Thirty-One as opportunities to expose the organization or to shut it down. (DS9: "Inquisition").
In late 2375, Agent Sloan appeared to Bashir again, assigning him to attempt to diagnose the health of Koval, the Chairman of the Tal Shiar, during an upcoming conference on Romulus, the Federation's new ally in the war against the Dominion. In the course of his attempts to expose Thirty-One, Bashir discovered that he had actually been manipulated by Sloan into revealing to Romulan Senator Cretak his suspicion that Section 31 planned to assassinate Koval in order to manipulate Cretek into attempting to access Koval's personal database. This gave Koval sufficient evidence to have Cretek removed from office, and placed Koval onto the powerful Continuing Committee, a seat upon which he was competeing with Cretek for. Bashir later learned that Koval was an agent of Section 31, after discovering Starfleet Admiral William Ross' complicity in the scheme to place Koval onto the Continuing Committee.
Section 31, it seemed, had been planning for what it regarded as a likely war with the Romulan Star Empire to follow the Dominion War; the agency regarded the Dominion as being likely to end up confined to the Gamma Quadrant, the Cardassian Union as likely to be left in shambles, and the Klingon Empire as likely to spend at least ten years rebuilding from the damage inflicted upon it by the Klingon-Cardassian War, the Klingon-Federation War of 2373-2374, and the Dominion War. (DS9: "Inter Arma Enim Silent Leges").
Later that year, while researching the disease ravaging the Great Link and Odo, Bashir discovered that Section 31 had artificially engineered the virus and infected Odo during his time on Earth in mid-2372—a pre-emptive attempt on Thirty-One's part to commit genocide. Bashir lured Sloan to DS9 with false claims of having developed a cure; there, he used an advanced technological procedure to link his mind and that of Chief Operations Officer Miles O'Brien to Sloan's, allowing Bashir to discover the real cure. Sloan later committed suicide, nearly killing Bashir and O'Brien (who were still linked to Sloan's mind) in the process. (DS9: "Extreme Measures"). The Federation Council later voted to refrain from sharing the cure with the Great Link, even while Odo was treated. This appears to have been part of a systematic attempt to cover up Section 31's existence—Bashir's and Sisko's 2374 reports had prompted the creation of an inquiry at Starfleet Command that was quietly stonewalled within one year, indicating that someone at Command was protecting Thirty-One. The cure was later given to the Founders by Odo in return for the surrender of the Dominion. (DS9: "What You Leave Behind").
- Although the televised appearances of Section 31 have been limited to Star Trek: Deep Space Nine, a short series of novels has also been published, documenting encounters with Section 31 in the other Star Trek series.