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Background/Objectives: The ocular surface is continuously exposed to microorganisms, and disruption of host–microbial balance may lead to infection or postoperative complications. Increasing antimicrobial resistance and biofilm formation have highlighted the need for alternative or complementary non-antibiotic strategies to control ocular surface microbial burden. Liposomal ozonated oil eyedrops have demonstrated antimicrobial and antibiofilm activity in preclinical and preliminary clinical studies. The aim of this study was to evaluate changes in ocular surface microbiological culture results before and after treatment with liposomal ozonated oil eyedrops in a real-world clinical setting. Methods: This was a prospective, observational, real-world pre–post study including 101 eyes from 101 patients undergoing ocular surface microbiological sampling in routine clinical practice. Two samples were obtained per patient: Sample I immediately before treatment and Sample II at the routine follow-up visit after short-course treatment with liposomal ozonated oil eyedrops (1 drop, four times daily, for 4 days). The interval between samples ranged from 3 to 5 days (median 3 days). Microbiological cultures were classified as positive or showing no growth. Paired changes in culture positivity were analyzed using McNemar’s exact test. Results: At baseline, 87 of 101 samples (86.1%) yielded positive cultures, while 14 (13.9%) showed no growth. Following treatment, culture positivity decreased to 11 of 101 samples (10.9%), with 90 samples (89.1%) showing no growth. Among baseline-positive samples, microbiological clearance was observed in 76 cases (87.4%). No cases converted from culture-negative to culture-positive at follow-up. The reduction in culture positivity after treatment was statistically significant (McNemar’s exact test, p < 0.001). Recent antibiotic exposure within 14 days prior to baseline sampling was reported in 8 patients (7.9%). Persistent positive cultures were observed in a minority of cases and were mainly associated with common ocular surface pathogens. Conclusions: In routine clinical practice, short-term treatment with liposomal ozonated oil eyedrops was associated with a significant reduction in ocular surface culture positivity over a short follow-up interval. Full article