The Joint United Nations Program on HIV/AIDS estimates that between 0.3% and 0.7% of adults aged 15 to 49 years were living with HIV in Ecuador in 2013. However, very little is known about the HIV prevalence rate among men who have sex with men (MSM) in that country. A cross-sectional survey was conducted to investigate the knowledge, attitudes, and practices regarding HIV/AIDS as well as to estimate the prevalence of HIV among MSM in one of the cities with high HIV prevalence rates in Ecuador. In this study, questionnaires were administered to 307 adult MSM. An HIV prevalence of 10% was observed. Knowledge about HIV was high; 91% of participants could identify how HIV is transmitted. Although consistent condom use for anal sex was relatively high (89%) among participants who reported having pay-for-service clients, only 64% reported using a condom during oral sex with a client. Participants who had multiple male sexual partners (i.e., their stable male partners plus other partner[s]) had 3.7 times higher odds of testing positive for HIV compared with those who did not. They also had reduced odds of condom use. Participants who were forced to have anal receptive sex had 3 times higher odds of testing positive for HIV. Despite the finding that participants exhibited high knowledge about HIV/AIDS, a high prevalence rate of HIV was observed, which warrants targeted behavioral interventions. These data are consistent with MSM being one of the highest at-risk population groups for HIV in this region of Ecuador.