The removal of 17β-estradiol (E2) by white-rot fungus Phanerochaete chrysosporium cultured in classic Kirk or potato medium was systematically investigated. Results demonstrated that E2 can be efficiently removed regardless of culture media type. However, the reaction intermediates and transformation pathways varied in different media. Estrone (E1) and estriol (E3) were sequentially generated as intermediates in the potato medium, but these intermediates were absent in Kirk medium. Such results were found to correlate to the peroxidases produced in Kirk medium. These enzymes catalyzed one-electron oxidation of E2 to form radicals that can undergo oxidative coupling. Similar enzymes were not detected in the potato medium, thus E2 underwent in vitro oxidation to form E1 and E3 sequentially. Adding glucose to the potato medium further accelerated such processes. The findings in this study provide insights into estrogen reactions mediated by P. chrysosporium and for potential development of biodegradation methods to reduce estrogen contamination levels.