Prisons are considered high-risk environments for HIV transmission. This study aimed to measure HIV and syphilis prevalence and risk behaviors among inmates in Indonesia. An integrated HIV and syphilis biological and behavior survey was conducted on random samples of 900 male and 402 female inmates in 2010. Male inmates from 18 general prisons and detention centers were randomly selected using probability proportional to size. Female inmates were randomly selected from nine eligible institutions. HIV tests included two rapid tests and enzyme-linked immunosorbent assay (ELISA) for quality control. A rapid test was used for syphilis. Audio computer-assisted self-interview was used for collecting risk behavior information. HIV prevalence was 1.1% among male and 6.0% among female inmates. Syphilis prevalence was 5.1% for male and 8.5% for female inmates. A history of injecting drugs was the most important risk factor for HIV infection in male inmates; for females, it was co-infection with syphilis and being sentenced for illicit drug use. Inmates’ high-risk activities in prison included tattooing, piercing and inserting genital accessories without sterile equipment, and sex without condoms. The study found high-risk practices by male inmates and high HIV and syphilis prevalence in female inmates. Inmates need harm reduction initiatives.