Bacteria are the most common causative agents of ocular infections. Treatment with topical broad-spectrum antibiotics is recommended in severe cases. However, antibiotic resistance has become a major concern in recent years, although antibiotics are generally effective in treating ocular infections. Antibacterial compound screening is performed to identify alternative therapeutic options to antibiotics. The aim of this study was to assess the in vitro antimicrobial activity of an ophthalmic solution containing ozonated oil. Strains of bacterial species with a multidrug resistance profile, which are responsible for a large proportion of ocular infections, were isolated and selected from different biological samples. The bacterial isolates were cultured, and ozonated oil was used to evaluate the inhibition zones at different time points. The treatment exhibited antibacterial activity against all the tested species. The effect was lower against the strains of Pseudomonas aeruginosa and more evident against Staphylococcus and Streptococcus spp. Our results suggest that the administration of ozonated oil may be a candidate agent to treat some infections of the ocular surface with a potential role in antimicrobial prophylaxis.