The use of intravaginal sponges produces changes in the composition of the normal vaginal microbiota and provokes the presence of a putrid abnormal fluid at sponge withdrawal. The aim of this study was to compare the effectiveness of the application of three different antibiotics (enrofloxacin, oxytetracycline or nitrofurazone) administered at sponge withdrawal on the vaginal microbiota in ewes. Intravaginal sponges impregnated with fluorogestone were inserted in 40 Pelibuey ewes (Day 0), remaining in situ for 14 days. At sponge withdrawal ewes received: 1) enrofloxacin, 2) oxytetracycline, 3) nitrofurazone, or 4) no treatment (control). The vaginal pH and temperature were recorded, and samples from the vaginal fluid were collected at that moment and 56 h later (Day 16). Samples were incubated to determine the number of Colony Forming Units (CFU). The log CFU increased from Day 0 to Day 14 (P < 0.0001) and decreased on Day 16 (P < 0.0001). On Day 16, the number of CFU was greater in control ewes than in the other three groups (P < 0.0001 for all the comparisons), being enroflaxin the more effective one (vs Toxi: P = 0.019 and vs Tnitro: P = 0.039). We concluded that the growth of the vagina microbiota present the day in which ewes come into estrus provoked by the use of intravaginal sponges may be partially controlled by the application of antibiotics at sponge withdrawal. Enrofloxacin was the most effective antibiotic to decrease bacterial vaginal number.