A cross-sectional study was conducted in 151 (71.6%) of 211 male inmates of a regional Portuguese prison in order to establish the seroprevalence for viral hepatitis (HAV, HBV, HCV), human immunodeficiency virus (HIV), syphilis and herpes simplex virus (HSV-1 and HSV-2) and to analyze some psychosocial and criminal characteristics. Mean age was 34 years. Anti-HAV was positive in 69.5% (n = 105) and in 34.4% (n = 52) for anti-HCV. One (0.7%) person had HBsAg and 29 (19.2%) had laboratory markers of past HBV infection. Non-immune inmates for HBV were 40.4% (n = 61). Syphilis was diagnosed in 6.0% (n = 9). The rate of HIV infection was 6.6% (n = 10; all HIV-1). The seropositivity of HSV-2 was 19.9% (n = 30) and of HSV-1 was 82.1% (n = 124). Alcohol dependence was reported by 26.5% (n = 40). Excluding tobacco and prescription medication, 73.5% (n = 111) reported drug use in prison. The most commonly used drugs were: cannabis (100%; n = 111) followed by heroin (56.7%; n = 63). Anti-HCV rate was noteworthy. The HIV infection rate (6.6%) in this regional prison is at least 13 to 22 times greater than in general population. As the inmate return to the community increases the risk of disease exposure for the general population, early detection and counseling is urgently needed for prisoners.