Infection of male urogenital tract or male accessory glands is considered as one of the important causes of male infertility, and results in the presence of bacteria in semen affecting fertility potential of men. It is important to know the composition of bacteria in semen to understand the etiology of urogenital infections and its association with infertility. This study aims to know the rate of infection in semen of infertile men, and the association of seminal bacteria with semen parameters related to fertility potential. A cross-sectional study was carried out from June 2021 to July 2022, in which 213 semen samples were collected from the male partners of couples consulting for fertility problems in an infertility center of Nepal. All the samples were processed following WHO guidelines, 2021. Analysis of semen parameters was done immediately after the liquefaction of collected samples. Microbiological assessment was also done for identification of bacteria in semen by conventional method, which showed 25.4% of samples had bacteriospermia. S. aureus and Corynebacterium were predominant bacteria in semen. The volume of semen was significantly associated with bacteriospermia. The concentration of sperms, percentage of total sperm motility, sperms with normal forms and vitality were found to be less in semen with bacteria compared to those without bacteria, which was statistically insignificant. This study provides a baseline data on bacterial infection in semen of infertile men in Nepal.