Background and objective <p>Lactic acid bacteria (LAB) are widely recognized for their probiotic and antimicrobial potential in food and health-related applications. This study aimed to isolate and characterize <i>Lactobacillus helveticus</i> strain CMI1 from traditional Iranian Khiki cheese and to evaluate its probiotic-related properties, including antimicrobial, anti-biofilm, antioxidant, and cytotoxic activities.</p> Methods <p>The isolate was identified by 16S rRNA gene sequencing. Antimicrobial activity of acidic and neutralized cell-free supernatants (CFS) was evaluated against major Gram-positive and Gram-negative foodborne pathogens using disk diffusion, well diffusion, modified double-layer, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. Anti-biofilm activity against <i>Staphylococcus aureus</i> was assessed using crystal violet staining and quantitative real-time PCR analysis of biofilm-related genes. Antioxidant activity was determined using DPPH, ABTS, and β-carotene/linoleic acid assays. Cytotoxic effects were evaluated against HT-29, HeLa, and MCF-7 cancer cell lines using the MTT assay. Antibiotic susceptibility was also examined.</p> Results <p><i>L. helveticus</i> CMI1 exhibited strong antimicrobial activity, with higher efficacy observed for acidic CFS compared to neutralized CFS, particularly against <i>S. aureus</i>. The strain significantly inhibited <i>S. aureus</i> biofilm formation and downregulated key biofilm- and virulence-related genes (<i>mecA</i>,<i> agr</i>,<i> spa</i>,<i> icaA</i>, and <i>hla</i>). Additionally, CMI1 demonstrated notable antioxidant activity and dose-dependent cytotoxic effects against cancer cell lines, while remaining sensitive to clinically relevant antibiotics.</p> Conclusion <p>These findings indicate that <i>L. helveticus</i> CMI1 possesses promising probiotic and functional properties, supporting its potential application in food preservation and functional food development.</p>