In more than half of infertile men, the cause of their infertility is unknown. Several studies revealed the role of viral infections in male infertility. The aim of the present study was to determine the prevalence of herpes simplex virus-1 (HSV-1) and HSV-2 in semen from asymptomatic infertile male patients, and its association with altered semen parameters. A total of 70 semen samples were collected from infertile men who attended the Research and Clinical Center for Infertility in Yazd, Iran. Semen analysis and diagnostic real-time PCR using specific primers and probes for HSV-1 and HSV-2 DNA were performed. Comparison of semen parameters between virally infected and non-infected samples were performed with independent t-test and Mann-Whitney test. Semen analysis showed that infertile men fell into two groups, the male factor group and the unexplained group. HSV-1 and HSV-2 DNA was detected in 16 (22.9%) and 10 (14.3%) of 70 semen samples, respectively. All HSV-positive samples had abnormal semen parameters (the male factor group). Although HSV infection was not associated with sperm motility and morphological defects, it was correlated with lower sperm count in the seminal fluid. The findings suggest that asymptomatic seminal infection of HSV plays an important role in male infertility by adversely affecting sperm count.