Background and aim: The escalating prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) has become a major public health threat. While lactobacilli were recently found useful in fighting various pathogens, limited data exist on their therapeutic potential for S. aureus infections. The aim of this study is to determine whether Lactobacillus Cell Free Supernatants (LCFS) were able to inhibit the biofilm produced by S. aureus. Material and methods: A total of 36 Staphylococcal bacteria were isolated from infected skin's patients. Antimicrobial susceptibility pattern of all tested isolates were determined using disk diffusion method. Biofilm production ability was detected using the Congo Red Agar (CRA) and the Microtitre Plate (MtP) assays. Furthermore, the efficacy of Lactobacillus CFS in inhibiting the biofilm skin infections induced by S. aureus isolates was estimated using MtP method. Results: Out of 36 S. aureus isolates, 34 (94.4%) and 20 (55.5%) showed positive results using CRA or MtP, respectively. All isolates showed high resistance against cefepime (97%), whereas imipenem was the most effective antibiotic against bacterial isolates. CFS exhibited significant anti-biofilm activities against the tested isolates. Conclusion: CFS of Lactobacillus spp. presents potential and safe alternative to synthetic antibiotics for inhibiting the biofilm skin infections induced by S. aureus.