The effect of bactericide Sekusept active (B SA), a peracetic acid-based preparation, on microbial strains, isolated from patients with severe infectious diseases who were treated in a regional children’s multi-specialty hospital, was studied. Based on the biochemical identification, the strains were classified as gram-negative non-fermenting bacteria (22 strains), Enterobacteriaceae family (18 strains), and bacilli - 3 strains. The biocidal activity of B SA was evaluated by the degree of inhibition of the growth of bacterial cells, existing in the planktonic form and in the form of biofilm (on a flat-bottomed plastic immunological tablet). It was shown that all the studied strains had the ability to biofilm formation, most of them (67,4%) formed moderately pronounced biofilms, and non-fermenting bacteria had a particularly pronounced coefficient of biofilm formation. The selected concentrations of B CA inhibited the growth of planktonic cells, and the ability of bactericide to prevent the formation of biofilms depended on the concentration (the most effective concentrations were 0,8 and 3,0%). Sensitivity of the strains existed in the aged biofilm to the bactericide was significantly lower, especially resistant to this effect were biofilms formed by non-fermenting bacteria and representatives of fam. Enterobacteriaceae. Our results confirm the importance of testing the effectiveness of biocides not only in accordance with standard methods developed for microorganisms in planktonic form, but also for biofilms.