OBJECTIVE To elucidate the association between asymptomatic infections caused by Mycoplasma genitalium and male infertility, and evaluate the role of antibiotic therapy in treatment of this failure. METHODS A total of 165 infertile males having abnormal semen parameters (study group) and 165 healthy fertile men (control group) were included. Semen samples were taken from all participants and after analyzing for semen parameters, undergone real-time PCR, microbial culture, and reactive oxygen species (ROS), as well as total antioxidant capacity (TAC) assays. Infected individuals of study group were treated with antibiotic. One month after the treatment completion, second semen samples were taken and subjected to all the tests mentioned. The data were analyzed using SPSS statistical software, version 22.0. RESULTS The frequency of M. genitalium was significantly higher in the infertile men compared with the fertile ones (9.7% vs. 1.2%; p = 0.001). Mean cycle threshold (C t) value was lower in infected infertile than infected fertile men (p < 0.001). All semen parameters, except volume, pH, and viscosity, were improved (p < 0.05), most of which reached their normal range; leukocytes in seminal fluid decreased (p = 0.02), the level of TAC was elevated (p = 0.002), and ROS level as well as ROS/TAC ratio reduced after antibiotic treatment (p = 0.03). Wives of seven infected infertile men (43.8%) became pregnant 4 months after the treatment completion. CONCLUSIONS Asymptomatic infection caused by M. genitalium is correlated with male infertility and antibiotic therapy can improve the semen quality and be used to treat male infertility.