The development of antibioﬁlm strategies is of major interest in contrasting bacterial bioﬁlms that are a predominant microbial style of life in natural and technical ecosystems. Тhe aim of this study is to evaluate the impact of metabolites produced during the cultivation of lactobacilli in MRS broth, on the biofilm-formation activity of co-cultures Bacillus subtilis and Escherichia coli K-12 strains. For this purpose, several classical microbiological tools, in particular method for static cultivating of biofilms in 96-well polystyrene plates, and confocal laser scanning microscopy were applied. Thus, the inhibitory effect of eight Lactobacillus strains, isolated from homemade dairy products manufactured in Rodopi Mountain, Bulgaria, has been estimated. A strain-specific anti-biofilm... activity of cells-free supernatants from eight exponential Lactobacillus cultures on the biofilms formed by Bacillus subtilis NBIMCC 170 and Escherichia coli K12 strain 1655 and by co-culture of B. subtilis NBIMCC 168 - E.coli K12- 1655 was observed. Lactobacillus plantarum L32 strains exhibited a good antibiofilm activity against co-cultures of Bacillus subtilis and Escherichia coli K12 strains. Data shows that the cell-free supernatants of Lactobacillus delbrueckii subsp. bulgaricus strain stimulate sporulation process in the structure of the biofilm by B.subtilis 170 and E.coli K-12 1655 strains and by B. subtilis 168 and E.coli K-12 1655 strains in comparison to Lactobacillus plantarum strains. In the structure of formed biofilms, the role of dominant species is implemented by B. subtilis strains in the presence of cell-free supernatants of Lactobacillus strains and at delution rate of cell-free supernatants of Lactobacillus plantarum L32 strains in MRS broth in the range from 1:10 to 1:1000. The data of the confocal laser scanning microscopy shows that at dilution rate of cell-free supernatant of 10-1 leads to appearance of blank optical field, the increase of metabotnite products of Lactobacillus plantarum L32 strain at dilution rate in the range of 10-2 – 10-3 creates conditions for increasing of intensity of staining by immunofluorescence days in this study. The obtained results showed that a strong anti-biofilm forming effect was obtained with Lactobacillus plantarum L32 culture in MRS broth.