This study investigated the potential of aquaculture to spread non-native species associated with a high risk of invasiveness. We identified the biogeographic origin of produced fish species in the micro-watershed of the São Camilo stream, Brazil, the occurrence of non-native species in the São Camilo stream and the potential risk of biological invasions. Fish farmers were interviewed to obtain information about the species produced, and samples with electrofishing equipment were taken in the stream to detect the occurrence of non-native species. The Fish Invasiveness Screening Kit (FISK) protocol was applied to classify non-native species according to their invasive potential. We identified 59 fish farms and 19 produced fish species, including 13 non-native species. The non-native species Oreochromis niloticus represented 93% of production and was the second most abundant species in the stream. Eleven species were classified with a high invasive potential and two with a medium potential. The results showed the prevalence of non-native species use in aquaculture, with species escapes with a high invasive potential. Thus, the non-native species produced in aquaculture represent an important vector of introductions and risk to biodiversity conservation. It is vital to establish biosafety norms, to enable aquaculture to develop in a sustainable way.