17β-Estradiol (E2) is a natural estrogen produced by the feminine endocrine system. It is excreted mainly through urine and feces. Exposure to E2 may affect the reproductive system of both animals and humans, especially since the removal of E2 in conventional processes and technologies present in the wastewater treatment plants is not sufficient. Chlorine is one of the most studied and used oxidant worldwide. Although there are studies that demonstrate the endocrine disrupting compounds removal like E2, its reaction with organic matter can originate by-products, namely, trihalomethanes, which are known to have high toxic potential. The main aim of the present study was to evaluate the removal of E2 (50 μg E2 L−1—maximum concentration) using peracetic acid (PAA), a seeming cleaner and innocuous alternative to chlorine. To this end, a series of jar tests were performed, using different peracetic acid concentrations (1, 5, 10, and 15 mg L−1) and contact times (10, 15, and 20 min). The results obtained showed that a peracetic acid concentration of 15 mg L−1 with a contact time of 20 min had a removal efficacy of approximately 100%. The second main goal of this study was to evaluate the ecotoxicological potential of the tested treatments on the zebrafish Danio rerio. Several oxidative stress biomarkers were evaluated, namely glutathione S-transferase, lipid peroxidation, and catalase, besides vitellogenin. Both peracetic acid and E2 caused significant increases in the oxidative stress biomarkers, although this did not lead to increased lipid peroxidation levels. In addition, peracetic acid significantly decreased the estrogenic activity of E2, as indicated by decreased vitellogenin levels. Peracetic acid demonstrated to have great potential as an alternative disinfectant for chlorine treatments, and indications for future research are discussed.