The natural estrogen 17beta-estradiol (E2) is a major endocrine disruptor, with adverse effects on wildlife and humans. The aim of this study was to isolate microorganisms able to effectively remove E2 from wastewater. Accordingly, five E2-degrading strains of bacteria were isolated from activated sludge collected from a wastewater treatment plant. Based on their 16S RNA gene sequences, these five strains belonged to the genus Bacillus. All five isolates were capable of converting E2 to estrone (E1), greatly reducing total estrogenic activities in wastewater during E2 biodegradation. However, only two strains (strain E2Y1 and E2Y4) were able to further transform E1, whereas it accumulated in the culture medium of the other isolates. Among all isolates, strain E2Y4, with 100% of the 1,400 bp 16S RNA gene matched that of B. subtilis CICC10075, exhibited the highest E2 and E1 degradation capacities, degrading 1 mg E2/l completely within 4 days and further transforming 40% of the metabolite E1. Furthermore, the E2 degradation rates of strain E2Y4 increased with increasing initial concentrations of the steroid, with a high degradation capacity maintained even at initial concentrations up to 50 mg/l. These results demonstrate the potential significance of strain E2Y4 in biological remediation applications.