Aims: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus , known as a causative agent of maternal breast infections and neonatal infections. Methods and Results: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus . Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis , Strep. salivarius , and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis ), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus , Lact. crispatus , Lactococcus lactis , Leuconoctoc mesenteroides ), isolated from 12.5% of samples, were also effective against Staph. aureus . One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. Conclusions: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus . Significance and Impact of the Study: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.