Diuron, an additive biocide in antifouling paints, is widely employed to curtail the attachment of organisms on submerged surfaces in aquatic structures. Despite the detection of diuron in aquatic ecosystems, information regarding its acute and chronic impacts on aquatic invertebrates, particularly planktonic crustaceans, remains limited. In this study, we analyzed the acute (24 h) and chronic (12 days exposure across three generations) effects of different concentrations of diuron (1/10 of the no observed effect concentration (NOEC), the NOEC, and 1/10 of the lethal concentration 50% (LC50), derived from the 24 h acute toxicity value of 1152 μg L−1) on the harpacticoid copepod Tigriopus japonicus. The acute exposure experiment indicated that the 1/10 LC50 value of diuron significantly reduced the copepod’s feeding rate and acetylcholinesterase activity. In response to the 1/10 LC50 value, the intracellular reactive oxygen species were elevated alongside increased malondialdehyde levels, while the glutathione content was depleted. The enzymatic activities of glutathione S-transferase, catalase, and superoxide dismutase were significantly enhanced by the 1/10 LC50 value, suggesting a proactive role of the antioxidant defense system against oxidative stress. Conversely, the activities of glutathione peroxidase and glutathione reductase enzymes were increased at the NOEC value, while their values were reduced by the 1/10 LC50 value. Chronic exposure to 1/10 NOEC and NOEC values revealed the adverse multigenerational effects of diuron. The second generation exhibited the most sensitivity to diuron, with the NOEC value notably reducing survival rate, body length, nauplius-to-adult development, neonates per brood count, and extending the reproduction period. Taken together, our findings underscore that even sublethal diuron levels can adversely impact copepod populations across generations through intergenerational toxicity.