This study applied life cycle assessment (LCA) to evaluate and compare environmental impacts of monoculture and polyculture systems in freshwater ponds. Two omnivorous native Brazilian species were used: the fish tambaqui (Colossoma macropomum) and the Amazon River prawn (Macrobrachium amazonicum). Four semi -intensive aquaculture systems (at an experimental level) were established and studied: monoculture of C. macropomum (MM), monoculture of M. amazonicum (MA), polyculture in which both species were free in the pond (PF), and polyculture in which C. macropomum was reared in a hapa cage and M. amazonicum was free in the pond (PH). The MM, PF and PH systems were fed fish feed, while MA was fed shrimp feed. Water was not renewed, but added only to replace losses from evaporation and seepage. Seven impact categories were analyzed: climate change, eutrophication, cumulative energy demand, land occupation, acidification, net primary production use and water dependence. Potential impacts of 1 kg of animal biomass produced by the systems were calculated, as was uncertainty in predictions based on uncertainty in data for the systems. Environmental impacts of each species in the polyculture systems were estimated using system expansion and different allocation approach: mass, energy and economic. PF and MM had the lowest impacts in all impact categories, while MA had the highest. When economic allocation was used, PF had lower impacts than MM per kg of C. macropomum. The rearing stage itself was the main contributor to eutrophication, land occupation and water dependence. However, feed was the main contributor to acidification and net primary production use in all systems. Only for PH was feed not the most significant contributor to climate change. Productivity and feed conversation ratio were key factors that defined the most efficient system from an environmental viewpoint. Our study demonstrated the advantage of rearing M. amazonicum in a polyculture instead of a monoculture, while no difference was found for C. macropomum. Changing the allocation approach revealed that aquaculture of M. amazonicum has lower impacts when the species is reared in polyculture systems. Moreover, aquaculture of native species remains in the early stages, and further development of its production chain may decrease its environmental impacts.