The Ivinhema River basin has experienced the greatest expansion of sugarcane cultivation in the state of Mato Grosso do Sul, Brazil. Theassessment of water quality through physical and chemical parameters and ecotoxicological studies, employing both bioindicators collected in situ and laboratory tests, provides more robust results for this region. In this context, the objective of this study was to analyze the composition and structure of the landscape around the sampling sites located in three streams (Vitória, Rosário, and Piravevê) belonging to the Ivinhema River basin to evaluate the impacts of sugarcane cultivation expansion on water quality and genotoxicity biomarkers in situ and ex situ using native fish species. Inthe sampling sites in the Rosário and Vitória streams, sugarcane is the main land use, while in the Piravevê stream, pasture areas represent the main land use. The acidic pH observed in the Vitória and Rosário streams seems to be related to the application of fertilizers and the fertigation in the sugarcane crop. Two species of detritivorous and five of omnivorous fishes were collected. A higher frequency of genotoxic damage was identified in detritivorous fish species. In the genotoxicity tests with Astyanax lacustris, the most frequent nuclear alteration induced by the water samples from all sampling sites was nuclear invagination. Our results indicated that the expansion of the sugarcane ridge in the Ivinhema River basin may cause negative impacts on the aquatic environment and native biota. These results contribute to the generation of data and information that can be used for public hearing requests that aim to discuss and review many aspects of legislation regarding agricultural activities around streams, as well as the need for restoration and management programs in these areas in order to conserve biodiversity.