Objective: The rise of antibiotic resistance necessitates exploring natural antimicrobial peptides like bacteriocins. This study aimed to investigate the anti-adhesive and anti-biofilm activities of acidocin A from Lactobacillus acidophilus against clinically relevant pathogens and its synergistic potential with nisin and human defensin HD-5. Methods: Recombinant peptides were obtained via heterologous expression in Escherichia coli. Anti-adhesive activity against Candida albicans was assessed on polystyrene using MTT assays and on Caco-2 monolayers by colony counting. Biofilm inhibition was tested against fungal and bacterial isolates, using resazurin and crystal violet, correspondingly. Synergy between the peptides was analyzed using checkerboard assays. Results and Discussion: Acidocin A inhibited C. albicans adhesion to polystyrene and Caco-2 model of intestinal epithelium. It effectively prevented biofilm formation by C. albicans and a number of bacterial strains, with inhibitory concentrations generally matching MICs. However, the peptide was inactive against mature biofilms. An additive effect of the peptide with human defensin HD-5 and acidocin 8912 was revealed. More importantly, acidocin A displayed synergism with nisin against E. coli (FICI 0.25–0.375), likely by permeabilizing the outer membrane to facilitate nisin entry. Conclusions: This study provides the first evidence of acidocin A’s anti-adhesive properties and efficacy in preventing biofilm formation by resistant pathogens. Its ability to synergize with nisin against Gram-negative bacteria highlights its potential for prophylactic and combination therapies.