<p>Diflubenzuron (DFB) is important to study due to its widespread use in agriculture and vector control. These harmful chemicals can persist at trace concentrations in aquatic systems for extended periods, potentially exerting adverse effects on aquatic life. This research investigated the toxic effects of DFB on the water flea (<i>Daphnia magna</i>) at environmental concentrations during a chronic exposure period of 21-days. <i>D. magna</i> is a key indicator species in freshwater ecosystems and plays a vital role in the aquatic food web. The EC₅₀ value was found to be 51.76 ng L⁻¹, with a 95% confidence interval (CI) of 45.65–58.70 ng L⁻¹. The survival rate of <i>D. magna</i> was 12.5% at a DFB concentration of 10 ng L⁻¹ during the chronic exposure period, which was significantly lower than that of the other groups (<i>p</i> &lt; 0.05). Moreover, the increased DFB concentration significantly reduced the fecundity of <i>D. magna</i>. In detail, the total of reproduction was significantly decreased according to increasing DFB concentration, while an increase in undeveloped embryos and male offspring was increased (<i>p</i> &lt; 0.05). Ultimately, the number of normal offspring capable of reproduction decreased, and the growth of organisms showed a concentration-dependent decline. Exposure to DFB induced a notable increase in the antioxidant enzymes superoxide dismutase and catalase, as well as lipid peroxidation in daphnids, suggesting an activation of the antioxidant system and cellular damage in organisms. Furthermore, chitin content increased in response to DFB exposure, suggesting a potential compensatory mechanism to strengthen the exoskeleton. These findings underscore the adverse effects of DFB on <i>D. magna</i> reproduction, growth, and overall physiological health, even at environmentally relevant concentrations. This research indicates the necessity of investigating the ecotoxicological risk of DFB contamination in aquatic ecosystems.</p>