Abstract: Bitter melon extracts were screened for antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Yeast, Penicillium and Aspergillus niger, the disc diffusion method was used to study the effect on antimicrobial activity of pretreatment temperature, extraction solvents, extraction methods to different bitter melon varieties (Guinongke-3, Bizhu-6 and Bite-111) , and two evaluation methods of antibacterial activity including disc diffusion method and oxford cup method were compared.Results showed that extracts obtained under the conditions of pretreatment temperature was 60℃, extraction solvent chose 70% ethanol, used ultrasonic assisted extraction had the strongest antibacterial activity.The antibacterial activity of bitter melon on Gram-positive bacterium was much better than Gram-negative bacteria, Yeast and Penicillium.The inhibitory effect on Staphylococcus aureus was the strongest, followed by Bacillus subtilis and the inhibitory effect on Escherichia coli and Yeast was weak.However, bitter melon had no effects on Penicillium and Aspergillus niger.To all the tested strains, the antibacterial activity of Bite 111 extractions was the best.The data of antibacterial activity measured by oxford cup method was more sensitive and better stability than the disc diffusion method.