Background. Pseudomonas aeruginosa is able to form a biofilm that protects cells under various conditions [1, 2]. Pyocins are one of the most widespread antagonism factors in P. aeruginosa [3]. These microorganisms synthesize various pyocins types with high activity and ability to prevent biofilm formation [4, 5]. Pyocins demonstrate nuclease activity [6], so they can perform a regulatory function. Aim. To assess the possibility of pyocins regulatory effect on P. aeruginosa. Methods. The activity of P. aeruginosa UCM B-333 pyocins was determined by the “double-layer agar” method [7]. Supernatants with pyocins were added in dilutions of 1:20—1:100 to suspensions of insensitive to them P. aeruginosa UCM B-2 and UCM B-9 with a titre of 1—2×10⁷ CFU/ml. Biofilm formation was studied for 3 days in 96-well plates, then stained with gentian violet, 96% ethanol was added, and adsorption was determined on a Multiscan FC at 620 nm (A620). Results. 1:20 pyocins dilution increased A620 of UCM B-2 by 3, 6, and 5.8 times compared to the untreated culture on 1, 2, and 3 days, and at a dilution of 1:50 — by 1.3; 2.6, and 2.8 times, respectively. Substances at a dilution of 1:100 did not change biofilm formation dynamics. The effect of 1:20 pyocins dilution on UCM B-9 was to increase in A620 by 3.7; 4.7 and 3.5 times during the entire observation period, and dilutions of 1:50 and 1:100 — by 1.5 and 1.1 times only on the first day, respectively. Instead, on 2 and 3 days, these dilutions reduced the A620 by 1.2—1.3 times. Conclusions. Pyocins at low dilutions can regulate the intensity of P. aeruginosa UCM B-2 and UCM B-9 biofilm formation.