Although infection with Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) frequently co-exist, there has been little research to determine the effects of HIV/HCV co-infection on health-related quality of life (HRQOL). We performed a cross-sectional analysis of baseline data from 216 participants enrolled in a community based study of HIV-infected homeless and marginally housed individuals, using multivariable linear regression analysis to determine if co-infection with HCV was independently associated with lower short-form 36 (SF-36) questionnaire scores. We found that individuals with HCV had significantly lower mean SF-36 scores in the domains of physical functioning, bodily pain, social functioning and role limitation due to emotional health, and that HIV/HCV co-infection was independently associated with a lower physical component score but not a lower mental component score after controlling for numerous covariates. These results suggest that co-infection with HCV may have an adverse effect on HRQOL among homeless and marginally housed individuals with HIV.