The increase of antibiotic resistance has become a problem. Probiotic bacteria play an important role in preventive/supportive medicine. Therefore, we examined the inhibitory effects of four different Lactobacillus species' (L. acidophilus-La, L. plantarum-Lp, L. fermentum-Lf and L. rhamnosus-Lr) cell-free supernatants (CFSs) on growth, adhesion, invasion, and biofilm formation of Staphylococcus aureus and effects of S. aureus, CFSs, and S. aureus-CFSs co-existence on human osteoblast (HOB) cell viability. Growth alterations were measured spectrophotometrically. Adhesive/invasive bacterial counts were detected by colony counting. Biofilm was evaluated using microtiter plate assay. The MTT assay was used for detection of HOB cell viability. The growth of MSSA significantly (P < 0.01) decreased in the presence of two CFSs (Lf and Lr) (P < 0.01); the growth of MRSA significantly (P < 0.05) reduced in the presence of La CFSs. All tested CFSs were found to reduce adhesion and invasion of MSSA (P < 0.0001). The adhesion of MRSA was enhanced (P < 0.0001) in the presence of all CFSs except La and the invasion of MRSA was decreased (P < 0.01) in the presence of Lr and Lf CFSs. All tested CFSs were shown to inhibit biofilm formation significantly (P < 0.0001). The reduction of S. aureus infected HOB cell viability and exposed to all CFSs except Lr that was found to be significant (P < 0.0001). The viability of HOB cell during co-incubation with MSSA and CFSs was shown to be decreased significantly. However co-existence of MRSA and CFSs did not alter HOB cell viability. These results suggested that lactobacilli as probiotics have low protective effects on MRSA-infected host cells.