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It has been proposed that Trametes versicolor laccase can be used to detoxify wastewaters that are contaminated with phenolic pollutants. However, the oxidation of phenols at low concentrations may be impacted if other substrates tend to interfere with or enhance the oxidation of the target substrate. To test this, experiments were conducted to evaluate effects arising from the simultaneous presence of mixed substrates including phenol (P), estradiol (E2), cumylphenol (CP), and triclosan (TCL), each of which are characterized by different rates of oxidation and tendencies to inactivate laccase. Slower and faster substrates were found to have only minor negative impacts upon the rate of conversion of targeted substrates, except where they tended to cause inactivation. No enhancements in substrate oxidation were observed. A multi-substrate kinetic model was shown to be able to accurately predict the time course of reactions of mixed substrates over extended periods at micromolar and sub-micromolar concentrations, except when estradiol and triclosan were simultaneously present. In this case, more enzyme inactivation was observed than would be expected from the oxidation of individual substrates alone. The utility of the model for providing insights into the reaction phenomenon and for evaluating the feasibility of oxidizing targeted substrates in the presence of other substrates is demonstrated.