The spread of multidrug-resistant (MDR) bacteria, such as the skin commensal Staphylococcus aureus, is a worldwide heath challenge; therefore, new methods to counteract the over-colonization and virulence of opportunistic pathogenic biotypes are highly urgent. We characterized and compared the activity of Lacticaseibacillus rhamnosus LR06 (DSM 21981) and Lactobacillus johnsonii LJO02 (DSM 33828) cell-free supernatants (CFSs), produced in a conventional animal-based MRS medium and in an innovative vegetal TIL, versus the MDR Staphylococcus aureus (ATCC 43300). CFSs were analysed via high-resolution mass spectrometry and gas-chromatography for short chain fatty acids (SCFAs), lactic acid and protein composition, while their activity was assessed towards i) the viability and metabolic activity of the MRSA strain through optical density and alamarBlue assay, and ii) the capability to inhibit/disaggregate the pathogenic biofilm, via crystal violet staining. All the CFSs reduce viable and metabolically active S. aureus, with the TIL medium more efficient, respect to MRS, in stimulating lactic acid bacteria metabolism and reducing the virulent biofilm. CFSs from LJO02 produced in TIL are the best, thanks to specific SCFAs and proteic metabolites. In conclusion, antagonistic non-pathogenic CFSs represent a promising and strategic approach, with potential applications as bacteriotherapy, and bioremediation of hospital equipments surfaces.