<p>Mixed bacterial endometritis poses a therapeutic challenge in mares due to the coexistence of multiple pathogens with divergent antimicrobial susceptibility profiles. This study evaluated the effects of intrauterine ozone insufflation on uterine bacterial load and endometrial inflammatory response in mares with naturally acquired mixed bacterial endometritis. Fifteen mares with mixed bacterial infection were allocated to two treatment groups: a pathological control group receiving pure medical oxygen insufflation (PC; <i>n</i> = 6) and an ozone-treated group receiving 42&#xa0;µg/mL ozone for three consecutive days (O3; <i>n</i> = 9). Eight clinically healthy mares subjected to the same ozone protocol served as a safety control group. Bacterial load was quantified in colony-forming units per milliliter (CFU/mL) from low-volume uterine flushings obtained at baseline (C1D0), six days post-treatment (C1D6), and at the onset of the subsequent estrous cycle (C2D0). A linear mixed model revealed significant group-by-time interactions for both bacterial load and neutrophil counts (<i>P</i> &lt; 0.001). At C1D6, the O3 group showed a median 100% reduction in bacterial load (from 65,000 to 0 CFU/mL; <i>P</i> &lt; 0.001), a culture positivity rate of 22.2% not significantly different from healthy controls (<i>P</i> = 0.260) and an 88.9% reduction in neutrophil counts. At C2D0, although bacterial cultures became positive again in all mares in the O3 group, bacterial load remained 77.5% below baseline (<i>P</i> &lt; 0.001), and neutrophil counts remained 42.4% below baseline, indicating a sustained biological response following treatment. The PC group showed no microbiological or inflammatory variation throughout follow-up. The healthy control group remained culture-negative at all time points. Intrauterine ozone insufflation substantially reduced bacterial load and endometrial neutrophil infiltration in mares with mixed bacterial endometritis, without inducing bacterial colonization in healthy animals.</p>