The mycelium of Lentinula edodes produces enzymes which may degrade xenobiotics including steroid hormones. The aim of the study was to determine whether the mycelium from in vitro cultures of L. edodes are able to degrade endocrine disruptors such as testosterone and 17α-ethynylestradiol. To prove the possibility of xenobiotics degradation, cultures of L. edodes were cultivated in an Oddoux liquid medium with the addition of synthetic 17α-ethynylestradiol and synthetic testosterone. The endocrine disruptors were extracted from the mycelium and determined qualitatively by RP-HPLC. The degradation products of testosterone and 17α-ethynylestradiol were identified using a UPLC/MS/MS analysis. Undegraded testosterone was determined at the amount of 2.97 mg/g dry weight but only in one of the L. edodes extracts from in vitro cultures supplemented with 50 mg of this compound. In turn, 17α-ethynylestradiol was not determined in any samples. Additionally in all extracts, mushroom sterols (ergosterol peroxide and ergosterol) were determined. Their total amounts were significantly lower in samples containing the abovementioned steroids than in extracts from mycelium L. edodes without the addition of steroid hormones. The results demonstrated that the mycelium of L. edodes can be used in the biodegradation process of a water environment contaminated with endocrine disruptors.