The risks posed by chemicals in the environment are typically assessed on a substance-by-substance basis, often neglecting the effects of mixtures. This may lead to an underestimation of the actual risk. In our study, we investigated the effects of three commonly used pesticides—imidacloprid (IMI), cycloxaprid (CYC), and tebuconazole (TBZ)—both individually and in combination, using various biomarkers to assess their impact on daphnia. Our findings indicated that the order of toxicity, from highest to lowest, was TBZ, IMI, and CYC, as determined by acute toxicity as well as reproduction. The effects of the ITmix (IMI and TBZ) and CTmix (CYC and TBZ) combinations on immobilization and reproduction were evaluated by MIXTOX, revealing a higher risk of immobilization at low concentrations for ITmix. The effect on reproduction differed depending on the ration of pesticides in the mixture, with synergism observed, which may be caused mainly by IMI. However, CTmix showed antagonism for acute toxicity, with the effect on reproduction depending upon the composition of the mixture. The response surface also exhibited a switch between antagonism and synergism. Additionally, the pesticides extended the body length and inhibited the development period. The activities of superoxide dismutase (SOD) and catalase (CAT) content was also significantly induced at different dosage points in both the single and combination groups, indicating changes in the metabolic capabilities of detoxifying enzymes and target site sensitivity. These findings highlight the need for more attention to be focused on the effects of pesticide mixtures.