We studied the effect of the L. plantarum strain supernatant on the growth of culture and biofilm of non-fermenting bacteria of the genera Pseudomonas, Achromobacter, and Burkholderia. To obtain a supernatant, the culture of L. plantarum was grown for 48 h at 37°C on a Lactic broth nutrient medium with casein peptone, then centrifuged and filtered through a 0.22-μm Millipore filter. Antimicrobial activity was determined by broth microdilution assay. The inhibitory effect of the supernatant on the growth of bacteria of all three genera was demonstrated. The maximum inhibition was observed for P. aeruginosa (by 13 times compared to the control). For bacteria of the Achromobacter and Burkholderia genera, the inhibition was less pronounced: by 7 and 6 times, respectively. The supernatant also inhibited biofilm formation by P. aeruginosa and A. ruhlandii, but did not affect formed biofilm. Thus, the L. plantarum supernatant obtained by us exhibited pronounced antimicrobial activity against non-fermenting bacteria, the causative agents of nosocomial infections, especially in immunocompromised individuals, very often in cystic fibrosis patients.