BACKGROUND The effects of cell-free culture supernatants of probiotic Lactobacillus rhamnosus GG and Streptococcus salivarius K12 on replication and biofilm forming of Staphylococcus aureus and S. epidermidis were assessed in vitro. METHODS S. aureus and S. epidermidis strains were exposed to cell-free culture supernatants of L. rhamnosus GG and S. salivarius K12 at different concentrations starting at 0, 4, and 24 h after the onset of incubation. Bacterial amplification was measured on microplate readers, as well as biofilm growth after safranine staining. Scanning electron microscopy was performed for visualization of biofilm status. RESULTS The S. salivarius K12 culture supernatant not only reduced or prevented the formation and maturation of fresh biofilms but even caused a reduction of preformed S. epidermidis biofilms. The L. rhamnosus GG culture supernatant did not show clear inhibitory effects regardless of concentration or time of addition of supernatant, and even concentration-depending promotional effects on the planktonic and biofilm growth of S. aureus and S. epidermidis were observed. CONCLUSIONS In particular, the inhibitory effects of the S. salivarius K12 culture supernatant on the formation of staphylococcal biofilms are of potential relevance for biofilm-associated diseases and should be further assessed by in vivo infection models.