Pesticides can cause harmful effects to aquatic communities, even at concentrations below the threshold limit established as guidelines for the water bodies by environmental agencies. In this research, an input of the herbicide 2,4-dichlorophenoxyacetic acid (i.e., 2,4-D) was simulated under controlled conditions in a 500-m-long reach of a first-order tropical stream in Southeastern Brazil. Two water samplings at eight stations investigated the stream longitudinal contamination profile. The ecotoxicological effects were analyzed using Eruca sativa L. seed germination assays and the acute and chronic toxicity tests with the neotropical cladoceran Ceriodaphnia silvestrii. Physicochemical parameters of water quality were evaluated to characterize the study area and quantify 2,4-D concentrations along the stream to assess pesticide retention. The 2,4-D concentration was reduced by approximately 50% downstream in the samplings, indicating that the herbicide was retained along the stream. Moreover, C. silvestrii reproduction in long-term assays decreased approximately 50% in the stations with higher concentrations of 2,4-D than the laboratory control. After contamination, E. sativa L. showed a lower average root growth (1.0 cm), statistically different from the control (2.2 cm). On the other hand, similar growth values were obtained among the background and the most downstream stations. Our study highlighted the relevance of reviewing and updating herbicide guidelines and criteria to prevent possible ecological risks.