This study aims to evaluate the antibacterial activity of Lactobacillus acidophilus, alone and in combination with ciprofloxacin, against otitis media-associated bacteria. L. acidophilus cells were isolated from Vitalactic B (VB), a commercially available probiotic product containing two lactobacilli species, L. acidophilus and Lactiplantibacillus (formerly Lactobacillus) plantarum. The pathogenic bacterial samples were provided by Al-Shams Medical Laboratory (Baqubah, Iraq). Bacterial identification and antibiotic susceptibility testing for 16 antibiotics were performed using the VITEK2 system. The minimum inhibitory concentration of ciprofloxacin was also determined. The antimicrobial activity of L. acidophilus VB1 cell-free supernatant (La-CFS) was evaluated alone and in combination with ciprofloxacin using a checkerboard assay. Our data showed significant differences in the synergistic activity when La-CFS was combined with ciprofloxacin, in comparison to the use of each compound alone, against Pseudomonas aeruginosa SM17 and Proteus mirabilis SM42. However, an antagonistic effect was observed for the combination against Staphylococcus aureus SM23 and Klebsiella pneumoniae SM9. L. acidophilus VB1 was shown to significantly co-aggregate with the pathogenic bacteria, and the highest co-aggregation percentage was observed after 24 h of incubation. The anti-biofilm activities of CFS and biosurfactant (BS) of L. acidophilus VB1 were evaluated, and we found that the minimum biofilm inhibitory concentration that inhibits 50% of bacterial biofilm (MBIC50) of La-CFS was significantly lower than MBIC50 of La-BS against the tested pathogenic bacterial species. Lactobacillus acidophilus, isolated from Vitane Vitalactic B capsules, demonstrated promising antibacterial and anti-biofilm activities against otitis media pathogens, highlighting its potential as an effective complementary/alternative therapeutic strategy to control bacterial ear infections.