Our previous research showed that the hydrophobicity of the extracellular polysaccharides produced by lactic acid bacteria plays an important role in biofilm formation on dairy separation membranes. Thermoduric bacilli possess a major challenge for the dairy industry, due to their resistance to heat and cleaning agents. The objective of this study was to evaluate the effect of slime production by Bacillus spp. on biofilm formation on separation membranes. Two slime-producing strains; Bacillus mojavensis (Bc) and Bacillus licheniformis (K1), isolated from dairy powder and one non-slime producing variant from each of the 2 strains (K1–1 from K1 and Bc-1 from Bc) produced by spontaneous mutation were used to study attachment (in the absence of growth) and biofilm formation on polyamide RO membrane pieces. Parameters related to bacterial adhesion (cell charge, capsule production, and hydrophobicity) were evaluated to determine their contribution to differences in biofilm formation among strains. The number of viable cells on biofilm formed by the hydrophobic Bc was more than 1 log cfu/cm² greater that its less hydrophobic slime-negative mutant (Bc-1) (P < 0.05). However, counts lower by about 0.7 log cfu/cm² were found in biofilm formed by the hydrophilic K1 than its slime-negative mutant (P > 0.05). Bc and K1 slime precipitated by ethanol contained only 3.6 and 6.5% of total carbohydrates. Bacterial cell surface hydrophobicity was the only parameter that strongly related to both attachment and biofilm formation on RO membranes. To confirm its role, cell surface hydrophobicity was modified by surfactants with different hydrophilic lipophilic balance (HLB) values and attachment of the altered cells was then studied. Tween 20 (high HLB) resulted in lower attachment of Bc while Span 80 (low HLB) improved biofilm formation by K1 (P < 0.05). In conclusion, hydrophobic slime produced by Bacillus enhanced attachment and biofilm formation on RO membranes. Cleaning strategies that decrease cell hydrophobicity or increase membrane hydrophilicity would reduce risk of biofilm formation by the potential spoilage and disease-causing Bacillus strains.