The conditions of aquatic environments have a great influence on the microbiota of several animals, many of which are a potential source of microorganisms of biotechnological interest. In this study, bacterial strains isolated from aquatic environments were bioprospected to determine their probiotic profile and antimicrobial effect against fish and food pathogens. Two isolates, identified via 16S rRNA sequencing as Lactococcus lactis (L1 and L2) and one as Enterococcus faecium 135 (EF), produced a bacteriocin-like antimicrobial substance (BLIS), active against Listeria monocytogenes, Salmonella Choleraesuis and Salmonella Typhimurium. Antimicrobial activity of BLIS was reduced when exposed to high temperatures and proteolytic enzymes (trypsin, pepsin, papain and pancreatin). All strains were sensitive to 7 types of antibiotics (vancomycin, clindamycin, streptomycin, gentamicin, chloramphenicol, rifampicin and ampicillin), exhibited a high rate of adherence to Caco-2 cells and expressed no hemolysin and gelatinase virulence factors. EF showed some resistance at pH 2.5 and 3.0, and L2/EF showed higher resistance to the action of bile salts. Finally, the presence of bacteriocin genes encoding for proteins, including Nisin (L1 and L2), Enterocin A, B, P, and Mundticin KS (EF) was detected. The molecular and physiological evidence suggests that the bacterial isolates in this study could be used as natural antimicrobial agents and may be considered safe for probiotic application.