Minimum Inhibitory Concentration (MIC) measures the lowest concentration of antibiotic that inhibits in-vitro growth of microorganisms. Canine pyometra is a frequently occurring reproductive disorder where reliable antibiotics along with surgical excision of uterus predicts better prognosis. Scarce literature is available to facilitate proper antibiotic selection to avoid septicemia in clinical cases. The present study was conducted to determine sensitivity and potency of selected antibacterials based on MIC values against bacterial isolates obtained from Canine Pyometra in Kathmandu district, Nepal by Agar Dilution method. Isolates obtained from 63 intra-uterine samples after ovariohysterectomy were Enterococcus spp (n=41), Klebsiella spp. (n=7), Staphylococcus spp. (n=11) and Streptococcus spp. (n=4). Enrofloxacin and Amikacin were found highly sensitive (0% resistance) with a null hypothesis of resistance to all bacterial isolates and were significantly more potent than other antibacterials tested (p