The ubiquitous presence of polystyrene nanoplastics (PSNPs) and di(2-ethylhexyl) phthalate (DEHP) in the aquatic environment may cause unpredictable negative effects on aquatic organisms and even continue to the offspring. This study assessed the transgenerational impacts of parental exposure to PSNPs and DEHP over four generations (F0-F3) of Daphnia magna. A total of 480 D. magna larvae (F0, 24 h old) were divided into four groups with six replicates (each of them contains 20 D. magna) and exposed with dechlorinated tap water (control), 1 mg/L PSNPs, 1 μg/L DEHP, and 1 mg/L PSNPs + 1 μg/L DEHP (PSNPs-DEHP) until spawning begins. Subsequent to exposure, all the surviving F1 offspring were transferred to new water and continued to be cultured until the end of F3 generation births in all groups. The results showed that the PSNPs accumulated in F0 generation and were inherited into F1 and F2 generations, and disappeared in F3 generation in PSNPs and PSNPs-DEHP groups. However, the accumulation of DEHP lasted from F0 generation to F3 generation, despite a significant decline in F2 and F3 generations in DEHP and PSNPs-DEHP groups. The accumulation of PSNPs and DEHP caused overproduction of reactive oxygen species in F0-F2 generations and fat deposition in F0-F3 generations. Additionally, single and in combination parental exposure to PSNPs and DEHP induced regulation of growth-related genes (cyp18a1, cut, sod and cht3) and reproduction-related genes (hr3, ftz-f1, vtg and ecr) in F0-F3 generations. Survival rates were decreased in F0-F1 generations and recovered in F2 generation in all treatment groups. Furthermore, the spawning time was prolonged and the average number of offspring was increased in F1-F2 generaions as a defense mechanism against population mortality. This study fosters a greater comprehension of the transgenerational and reproductive effects and associated molecular mechanisms in D. magna.