Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H2O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4-used as an indicator strain, B. licheniformis T6-5, and B. firmus H2O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H2O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H2O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H2O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.