Sugarcane crops management in Brazil includes the use of pesticides, as well as alternative organic fertilizers such as vinasse obtained from waste of the ethanol industry. In order to assess the effects of the environmental contamination generated by such sugarcane practices, this study was aimed to investigate the effects of the pesticides 2,4-Dichlorophenoxyacetic acid (2,4-D) and fipronil, as well as vinasse, on the survival, behavior, and reproduction of the native epibenthic macroinvertebrate Hyalella meinerti through in situ and laboratory experiments. In situ assays were conducted in mesocosms with six treatments, i.e. untreated control, 2,4-D, fipronil, and vinasse, the mixture of the two pesticides, and both pesticides mixed with vinasse. Survival, swimming behavior, and reproduction were evaluated over time post contamination, from 0-96 h (T1) and 7-14 days (T2) through in situ experiments and 30-44 days (T3) and 75-89 days (T4) post contamination by laboratory bioassays with mesocosm water. In the T1 period, survival of H. meinerti was registered only in controls and mesocosms treated with 2,4-D. In the T2 period, treatments containing fipronil and vinasse (isolated or in both mixture treatments) still caused 100 \% of mortality. Survival was recorded only in 2,4-D and control treatments, whereas reproduction only occurred in the control. In the T3 period, no survival occurred to fipronil and both mixture treatments. Vinasse and 2,4-D decreased total reproduction in comparison to control. In the T4 period, amphipods survival was detected when exposed to fipronil and its mixture with 2,4-D. However, these same treatments decreased the amplexus rates and total reproduction, with synergism denoted for the pesticide mixture. The swimming activity of males, females, and couples was decreased in surviving organisms exposed to 2,4-D, fipronil, vinasse, and the mixture of pesticides along all experimental periods. Our study showed that the application of fipronil, 2,4-D, and vinasse isolated or mixed at realistic concentrations of actual sugarcane management practices may negatively impact functional responses of indigenous amphipods in natural aquatic systems.