We studied the prevalence of HIV, syphilis, hepatitis B virus (HBV), and hepatitis C virus (HCV) infection and associated risk behaviors in the prison state system of Guanajuato, Mexico between September 2011 and February 2012. Blood samples were drawn from adult inmates in all State prisons who agreed to participate in this cross-sectional study. Data on risk behaviors were collected by using self-administered questionnaires. The prevalence of HIV, syphilis, HBV, and HCV infection was 0.6% [95% confidence interval (CI) = 0.2-1.1], 0.7% (95% CI = 0.4-1.0), 0.4 (95% CI = 0.04-0.74), and 4.8 (95% CI = 3.6-5.9), respectively. Female inmates had a higher prevalence of HIV (1.5% vs. 0.6%, p = .05), whereas male inmates had a higher prevalence of HCV (1% vs. 5%, p = .008). Twenty percent (n = 443, 95% CI = 15-26) of the participants were tattooed during incarceration, and most of them were tattooed with recycled materials. Around 60% (57%, 95% CI = 49-65) used drugs before incarceration, and 9.2% (n = 205) used injected drugs. During incarceration, 30% (95% CI = 23-39) used drugs and 43 continued injecting (20% of users). Consistent condom use was low among men before incarcerations but decreased by half during incarceration. The highest consistent condom use before incarceration was among men who have sex with men (MSM) (17.7%, 95% CI = 14-22), but it decreased (9%, 95% CI = 3-14) during incarceration. The prevalence of HIV, HBV, HCV, and syphilis in these inmates is higher than that of the local adult population. Most inmates had sex in prison, but few used condoms consistently. Access to condoms is apparently harder for MSM. Interventions to increase condom use, reduce use of shared or recycled materials for tattooing and injecting drugs, and treatment for drug abuse are needed.