There is a reemergence of syphilis in the Latin American and Caribbean region. There is also very little information about HIV/Syphilis co-infection and its determinants. The aim of this study is to investigate knowledge, attitudes, and practices regarding sexually transmitted infections (STIs), in particular syphilis infection and HIV/Syphilis co-infection, as well as to estimate the prevalence of syphilis among men who have sex with men (MSM) in a city with one of the highest HIV prevalence rates in Ecuador. In this study, questionnaires were administered to 291 adult MSM. Questions included knowledge about STIs and their sexual practices. Blood samples were taken from participants to estimate the prevalence of syphilis and HIV/syphilis co-infection. In this population, the prevalence of HIV/syphilis co-infection was 4.8%, while the prevalence of syphilis as mono-infection was 6.5%. Participants who had syphilis mono-infection and HIV/syphilis co-infection were older. Men who had multiple partners and those who were forced to have sex had increased odds of syphilis and HIV/syphilis co-infection. A high prevalence of syphilis and self-reported STI was observed, which warrants targeted behavioral interventions. Co-infections are a cause for concern when treating a secondary infection in a person who is immunocompromised. These data suggest that specific knowledge, attitudes, and behaviors among MSM are associated with increased odds of STIs (including HIV/syphilis co-infections) in this region of Ecuador.