Abstract: Objective To investigate the effect of semen bacterial infection on semen parameters in male infertility patients and analyze of the drug resistance profile of the bacterial isolates. Methods According to the results of bacterial culture, the semen samples of 74376 infertile men collected between April, 2016 and April, 2017 were divided into infection group and noninfection group, and the infertility type and semen parameters were compared between the two groups. The bacterial species and the drug sensitivity of the isolates were analyzed. Results Bacterial infections were detected in 1.38% of the total semen samples collected. The positivity rate of semen bacterial infection was 1.41% in normal semen group, 1.55% in asthenospermia group, 1.18% in oligospermia group, 1.57% in asthenospermia/ oligospermia group, and 0.17% in azoospermia group. The positivity rate was lower in azoospermia group than in the other groups. Bacterial infection mainly affected sperm motility (P< 0.05), sperm density and forward motile sperm ratio (P<0.01). The most common bacterial species causing the infections included, in the descending order of frequencies, Escherichia coli (63.59%), Klebsiella pneumoniae subspecies (19.80%) and Proteus mirabilis (13.22%). Drug susceptibility tests showed that the isolates of Escherichiacoli, Pneumonia Klebsiella pneumonia subspecies and Citrobacter koseri were commonly resistant to amoxicillin; Pseudomonas aeruginosa isolates were resistant to Bactrim and ampicillin/sulbactam; Staphylococcus aureus isolates were resistant to penicillin. Conclusion Male infertile patients have a low bacterial infection rate in the semen. Bacterial infection severely affects the sperm fertilization process by causing impairment of sperm motility and lowered sperm density. Escherichia coli is the most common pathogenic bacterium for the semen infection. With the exception of Staphylococcus aureus, the other bacterial strains isolated were found to be sensitive to imipenem and meropenem.