Fish farming is increasingly important globally and nationally, playing a crucial role in fish production for human consumption. Monitoring microbiological and chemical contaminants from water discharge is essential to mitigate the risk of contaminating water and fish for human consumption. This study analyzes the physicochemical and E. coli parameters of water and tambacu fish muscles (Colossoma macropomum × Piaractus mesopotamicus) in Western Maranhão, Brazil. It also includes a qualitative characterization of zooplankton in the ponds. Samples were collected from tambacu ponds in a dam system fed by natural watercourses from the Tocantins River tributaries, located at the connection of the Brazilian savanna and Amazon biomes. The physicochemical and E. coli parameters of water did not meet national standards. The zooplankton community included Rotifera, Cladocera, Copepoda, and Protozoa representatives, with no prior studies on zooplankton in the region, making these findings unprecedented. The biological quality of freshwater is crucial in fish farming, as poor quality can lead to decreased productivity and fish mortality, raising significant food safety concerns. The water quality studied is related to the potential influence of untreated wastewater as a source of contamination, leaving the studied region still far from safe water reuse practices. The findings on chemical and E. coli contamination of fish farming waters concern human health and emphasize the need for appropriate regulations.