Biofilm formation by pathogenic bacteria, including methicillin-resistant (MRSA), , and , represents a major clinical challenge due to the high resistance of biofilms to conventional antimicrobial therapy. In this in vitro study, we investigated the antimicrobial and antibiofilm activity of synthetic peptides R23I, R23L, V31K, R44K, R44K, V31K, and I31K against methicillin-resistant (MRSA, SA180-F strain), (129B), (2943), and (MG1655). In liquid medium, peptides R23L and R44K exhibited the broadest and most potent antimicrobial activity against all tested strains. On solid agar, these peptides demonstrated comparable activity, with notable effects particularly against . We further assessed the peptides' impact on both early-stage and mature biofilms using crystal violet staining for total biomass and the MTT assay for cellular metabolic activity. Peptide R44K showed a strong dose-dependent inhibitory effect on early MRSA biofilm formation, while most peptides unexpectedly enhanced biofilm formation by and . Peptides R44K and V31K at 10 mg/mL significantly reduced both biomass and metabolic activity of early biofilms. None of the peptides inhibited mature biofilm biomass across species; however, several, particularly I31K, significantly reduced the metabolic activity of MRSA within mature biofilms. These findings underscore the strain- and stage-specific effects of antimicrobial peptides and highlight R23L, R44K, R44K, V31K, and I31K as promising candidates for targeted biofilm control in vitro, especially against MRSA.