Introduction/Objective Biofilm and pyocyanin production are essential components of Pseudomonas aeruginosa virulence and antibiotic resistance. Our objective was to examine inhibitory effect of synthetized propafenone derivatives 3-(2-Fluorophenyl)-1-(2-(2-hydroxy-3-propylamino-propoxy)-phenyl)-propan-1-one hydrochloride (5OF) and3-(2-Trifluoromethyl-phenyl)-1-(2-(2-hydroxy-3-propylamino-propoxy)-phenyl)-propan-1-one hydrochloride (5CF3) on biofilm and pyocyanin in Pseudomonas aeruginosa clinical strains. Methods Effects were tested on nine clinical isolates and one control laboratory strain of P. aeruginosa. In vitro analysis of biofilm growing was performed by incubating bacteria (0.5 McFarland) with 5OF and 5CF3 (500-31.2 µg/ml) and measuring optical density (OD) at 570 nm. Bacteria in medium without compounds were positive control. Blank medium (an uninoculated medium without test compounds) was used as negative control. Pyocyanin production was estimated by OD at 520 nm, after bacteria incubated with 5CF3 and 5OF (250 and 500 μg/ml), treated with chloroform, and chloroform layer mixed with HCl. Results A total of 500 µg/ml of 5OF and 5CF3 completely inhibited biofilm formation in 10/10 and 4/10 strains, respectively. A total of 250 µg/ml of 5OF and 5CF3 strongly inhibited biofilm formation in 7/10 strains, while inhibition with 125 µg/ml of 5OF and 5CF3 was moderate. Lower concentrations had almost no effect on biofilm production. Pyocyanin production was reduced to less than 40% of the control value in 6/9, and less than 50% of the control in 7/9 strains with 500 μg/ml of 5OF and 5CF3, respectively. At 250 µg/ml 5OF and 5CF3, most strains had pyocyanin production above 50% of the control value. Conclusion Synthetized propafenone derivatives, 5OF and 5CF3, inhibited biofilms and pyocyanin production of Pseudomonas aeruginosa clinical strains. Presented results suggest that propafenone derivatives are potential lead-compounds for synthesis of novel antipseudomonal drugs.