Human-to-animal reverse transmission of SARS-CoV-2 is a risk for new reservoirs' emergence and new variants' evolution. SARS-CoV-2 infection of synanthropic rodents in urban settings has been reported during COVID-19 in New York and Mexico cities. In this study, we addressed the potential transmission of SARS-CoV-2 to synanthropic rats in the city of Guayaquil (Ecuador) during the COVID-19 pandemic. A total number of 234 rats were collected and analyzed for SARS-CoV-2 detection by RT-qPCR. A positivity rate of 6% (14 rats) was found, and SARS-CoV-2 infection was confirmed by Sanger sequencing of the viral genome. Our results confirm the potential risk of synanthropic rats as reservoirs for SARS-CoV-2 infection. This is worrisome for low and middle income countries like Ecuador, where pest and waste control in urban settings is challenging. Moreover, the risk of spillover to wild fauna is a concern in Guayaquil, where synanthropic fauna includes raccoons or coatis and forest patches with a wild population of felids or primates existing within the city limits. In this context, SARS-CoV-2 sentinel surveillance of synanthropic rodents could serve as a proxy for a One Health approach to prevent the emergence of new wild reservoirs.