Fish form important fisheries and aquaculture resources worldwide. In Brazil, pearl eartheater (Geophagus brasiliensis) is an important commercially exploited species and is an ideal animal for studying the impairment caused by the effects of herbicides that are often detected in the aquatic environment. The main purpose of the present study was to detect the acute toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) to G. brasiliensis and investigate its effects on oxygen consumption, ammonium excretion, and the neutral red retention time assay to estimate effects at the cellular level. Such investigations have not been carried out before with this species. First, the acute toxicity of 2,4-D to G. brasiliensis in terms of the 24-, 48-, 72-, and 96-h medium lethal concentration (LC₅₀) was calculated to be 45.95, 32.49, 28.28, and 15.16 mg/l, respectively. Furthermore, it was found that exposure of fish to 40 mg/l 2,4-D caused reduction in oxygen consumption and ammonium excretion of 59% and 85%, respectively, in relation to the controls. Mean neutral red retention time assay was significantly lower in comparison with control for organisms exposed to 1, 5, 10, and 40 mg/l 2,4-D. However, the effects at the cellular level were progressive, suggesting that the fish are not able to recover from such increasing effects.