Abstract A set of bioassays (acute toxicity to Daphnia, genotoxicity, estrogenic and antiestrogenic toxicity) was used to evaluate the safety of reclaimed water reuse in a lab-scale artificial groundwater recharge system. Ozonation of 1 mg O 3·(mg DOC) − 1 was used as the pretreatment and was proved to be effective in removing the four toxicity, with removal ratios ranging from 56% to 99%. In the vadose layer, the responsible compounds for toxicity were less efficiently removed than the bulk organic compounds. However, they were preferentially removed during the saturated aquifer treatment, with all the toxic effects per carbon mass dropping by 20–50%. Therefore, more than one month traveling in the aquifer is suggested before water reuse. Since UV 254 was significantly correlated with the toxicity data, it could serve as an indicator of the toxicity. With the bioassay-based safety evaluation, the combination of soil aquifer treatment and ozonation could provide new water sources with no higher toxic than the conventional natural drinking water source.