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In this study the enzymatic degradation of hormones and endocrine disrupting compounds (EDCs) was investigated in artificial mixtures and in real wastewater by fungal laccases (Trametes versicolor, Myceliophthora thermophila). Several studies have already reported the successful enzymatic degradation of EDCs. However, with regards to a large-scale application, the influence of some factors such as enzyme immobilization and costs are often lacking. Furthermore, the majority of studies investigated the removal of EDCs by chemical analysis only, while our main interest was to use bioassays to study the decrease in the endocrine activity. The removal of estrogenic, androgenic, and antiandrogenic activity was assessed by yeast-based reporter gene assays and the degradation of industrial chemicals by an additional chemical analysis. It was demonstrated that the degradation of hormones and EDCs by laccases is feasible even at very low enzyme concentrations (2.8 ABTS U/L). In the artificial mixtures the main removal mechanism was adsorption onto immobilization supports. In binary mixtures, immobilized laccase was best in removing EDCs within the first 6 h of exposure (83% for T. versicolor and 87% for M. thermophila), but in the course of the experiment adsorption superimposed this removal after 24 h (99% after 24 h). A similar pattern was seen in wastewater, but with less carrier material (lower adsorption) and a constant enzyme activity, immobilized laccase showed the best removal rates of estrogenic (82% removal after 24 h) and androgenic activity (99% removal after 6 h). With this in mind, this enzymatic technology can be a valuable addition to other treatment technologies.