Aquaculture is expected to be one of the most prominent source of animal proteins for humans in the next decades. Its importance for human food security has triggered many academic debates to improve its sustainability. Among potential solutions, polyculture of fish has previously been pointed out as a relevant alternative to monoculture in recirculated aquaculture system (RAS). However, previous studies assessed potential impacts of polyculture on a target species, and disregard the polyculture consequences on other taxa combined with this species. Yet, it cannot be ruled out that polyculture results in beneficial consequences for one species at the detriment of other taxa. Therefore, we developed a RAS polyculture design associating three fish species: pikeperch (Sander lucioperca), sterlet (Acipencer ruthenus), and tench (Tinca tinca). A total of 756 fishes was reared during 60 days and comprehensive analysis of polyculture consequences was performed for each of these three species by considering survival, growth, and behavioural parameters. All studied species were impacted by polyculture in our experimental design. However, species were not equally affected by polyculture: sterlet was more impacted in terms of survival, growth, and behaviour than pikeperch and tench. Therefore, our results suggest that RAS polyculture could result in beneficial or detrimental impacts on fish production depending on the species. Overall, this study underlines the importance of considering all species and a multi-criterion assessment framework to evaluate RAS polyculture.