Biofilms are highly important to be controlled in food industries for two major reasons: (i) pathogenic microorganisms can impact public health causing foodborne illness outbreaks, and (ii) food-spoilage microorganisms can cause economic impacts due to the loss of organoleptic quality. Listeria monocytogenes and Pseudomonas fluorescens are ubiquitous and highly representative of both problems. The presence of these bacteria in biofilms must be controlled, and new strategies need to be implemented. Among those strategies, the use of biosurfactants is promising. The present work studied the application of a surfactin produced by Bacillus velezensis H2O-1 to inhibit corrosion, planktonic growth, microbial adhesion, and biofilm formation by two strains of L. monocytogenes and one strain of P. fluorescens. For that purpose, scanning electron microscopy, epifluorescence microscopy, and the determination of the physicochemical characteristics of different surfaces, microorganisms and biofilms were performed. Biofilm reduction on conditioned surfaces reached up to 75%. When the surfactin was added to the media, the planktonic inhibition values reached 87%, and biofilms were inhibited by up to 100%. The analyzed images suggest that this molecule has great potential to postpone steel corrosion. The results demonstrated the great potential of this biomolecule in the food industry against both microorganisms, thus enhancing food safety and shelf-life.