The presence of pharmaceutical residues in the aquatic environment is receiving great attention since the levels of these substances have significantly increased in this compartment, potentially leading to adverse ecological effects. Zinc pyrithione (ZnPt) is a widely used organometallic biocide, which is incorporated into antifouling formulas, such as paints, to prevent the establishment of biofilms on surfaces exposed to the aquatic environment. It is also used in cosmetics, such as antidandruff shampoos and soaps. Considering this wide use, and the absence of a significant amount of data on the toxicity of ZnPt especially towards non-target organisms, the objective of this study was to characterize the toxicity of ZnPt, on several ecological relevant endpoints assessed in the fish Gambusia holbrooki. For this purpose, we measured traits related to feeding and aggressive behavior, as well as indicators of oxidative stress (CAT and GSTs), neurotoxicity (AChE), and anaerobic metabolism (LDH), after acute and chronic exposures to ZnPt. In terms of behavioral features, the feeding test showed the occurrence of significant differences between the control animals and those exposed to a concentration of ZnPt of 45 μg/L. In addition, ZnPt caused changes in terms of oxidative stress biomarkers (CAT and GSTs), for both exposure periods. ZnPt was also capable of causing changes in the cholinergic neurotransmission functioning and anaerobic metabolism, but only following the chronic exposure.