This study was undertaken to determine the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) among homeless persons with co-occurring severe mental illness (SMI) and substance use disorders and to determine associated risk factors. As part of a longitudinal study of the effectiveness of integrated treatment for homeless persons with SMI and substance abuse or dependence, serological testing was performed to ascertain the prevalence of HIV, HBV, and HCV. At baseline, 6.2% of participants (11/172) were HIV-positive. Nearly one third of participants (37/114) had evidence of prior exposure to HBV, and 30% (34/114) were antibody positive for HCV. About 44% of participants (50/114) had a reactive test for either HBV or HCV. Having a reactive test was strongly associated with substance use, especially with a history of injection drug use. A significant threat exists to the health and well-being of homeless person with SMI due to high prevalence of blood-borne pathogens. Mental health providers need to play a proactive role in the identification of health-related needs and to assist with access to general health services for persons with SMI.