This case report describes the isolation of Escherichia coli, Morganella morganii, and Staphylococcus spp from the vagina of a 3-year-old bitch presenting with chronic infertility and abnormal vaginal discharge. The bitch, which had whelped only once at 1 year and 5 months, experienced multiple failed breeding attempts despite an up-to-date vaccination status and treatment with amoxicillin before the last breeding. Two weeks post-breeding, a blackish vaginal discharge was observed, prompting further investigation. Microbial culture from a vaginal swab revealed three distinct organisms: E. coli as a lactose fermenter producing a greenish metallic sheen on EMB agar, Staphylococcus. spp as gram-positive cocci in clusters, and M. morganii as a non-lactose fermenter. Biochemical tests confirmed the identities of these isolates, and antimicrobial sensitivity testing indicated susceptibility of E. coli and M. morganii to gentamicin and streptomycin, while Staphylococcus spp was most susceptible to ciprofloxacin. Resistance to amoxicillin and other commonly used antibiotics was observed. Treatment was instituted using gentamicin and ciprofloxacin. Four weeks after treatment, a follow-up swab was collected from the bitch for culture, which yielded no growth after 48 hours suggesting that the infection had been cleared. The bitch was bred during the subsequent estrus and she successfully conceived and whelped 7 healthy puppies. This case highlights the significance of polymicrobial infections in canine reproductive health and emphasizes the role of targeted antimicrobial therapy guided by culture and sensitivity results, especially following previous antibiotic exposure.