Bacillus subtilisis traditionally classified as a PGPR that colonizes plant roots through biofilm formation. The current study focused on investigating the influence of various factors on bacilli biofilm formation. In the course of the study, the levels of biofilm formation by the model strainB. subtilisWT 168 and on its basis created regulatory mutants, as well as strains of bacilli with deleted extracellular proteases under conditions of changes in temperature, pH, salt and oxidative stress and presence of divalent metals ions.B. subtilis168 forms halotolerant and oxidative stress-resistant biofilms at a temperature range of 22 °C–45 °C and a pH range of 6–8.5. The presence of Ca2+, Mn2+and Mg2+upsurges the biofilm development while an inhibition with Zn2+. Biofilm formation level was higher in protease-deficient strains. Relative to the wild-type strain,degUmutants showed a decrease in biofilm formation,abrBmutants formed biofilms more efficiently.spo0Amutants showed a plummeted film formation for the first 36 h, followed by a surge after. The effect of metal ions and NaCl on the mutant biofilms formation is described. Confocal microscopy indicated thatB. subtilismutants and protease-deficient strains differ in matrix structure. The highest content of amyloid-like proteins in mutant biofilms was registered fordegU-mutants and protease-deficient strains.