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The aim of this study was to evaluate the antimicrobial and antibiofilm activity of <i>Weissella cibaria</i>, <i>Weissella hellenica</i> and <i>Bacillus coagulans</i>, isolated from equine skin, against biofilm-forming <i>Staphylococcus aureus</i> CCM 4223 and clinical isolate methicillin-resistant <i>S. aureus</i> (MRSA). Non-neutralized cell-free supernatants (nnCFS) of tested skin isolates completely inhibited the growth and biofilm formation of <i>S. aureus</i> strains and caused dispersion of the 24 h preformed biofilm in the range of 21–90%. The majority of the pH-neutralized cell-free supernatants (nCFS) of skin isolates inhibited the biofilm formation of both <i>S. aureus</i> strains in the range of 20–100%. The dispersion activity of <i>B. coagulans</i> nCFS ranged from 17 to 77% and was significantly lower than that of nnCFS, except for <i>B. coagulans</i> 3T27 against <i>S. aureus</i> CCM 4223. Changes in the growth of <i>S. aureus</i> CCM 4223 in the presence of catalase- or trypsin-treated <i>W. hellenica</i> 4/2D23 and <i>W. cibaria</i> 4/8D37 nCFS indicated the role of peroxides and/or bacteriocin in their antimicrobial activities. For the first time, the presence of the <i>fenD</i> gene, associated with biosurfactants production, was detected in <i>B. coagulans</i>. The results of this study showed that selected isolates may have the potential for the prevention and treatment of biofilm-forming <i>S. aureus</i> infections.