Various studies have investigated the effects of paracetamol on D. magna, with abundant information on acute and chronic toxicity, but little on multigenerational molecular-level assays. Therefore, this study is focused on a multigenerational investigation of the effects of paracetamol exposure at a constant concentration of 1 mg L− 1 on the expression of the cellular detoxification genes HR96 and GST in D. magna over three generations. The effective concentration of this compound was also determined by studying daphnid survival. The acute exposure results showed mean effective concentrations of EC50 = 11.74 mg L− 1 (11.07–12.48) and EC1 = 1.63 mg L− 1 (1.40–2.62). The multigenerational analysis showed a significant inhibition of both genes in both generation F0 (HR96 = 32.6%, GST = 33.2%) and generation F1 (HR96 = 27.1%, GST = 20.2%). Meanwhile, in F2 a significant induction was observed for GST and HR96, with values of 10.8% and 13.5%, respectively. The response of D. magna over the generations presented a clear induction trend, indicating that this organism is capable of adapting to the contaminant after multiple generations of exposure. Thus, this study shows that paracetamol is more toxic during the first generations and that after 30 days, the drug would not affect the cellular detoxification system.