1,4-Dioxane, a contaminant increasingly detected in water supplies, is a public health concern because it is classified as a possible human carcinogen. 1,4-Dioxane can be biodegraded by aerobic bacteria via monooxygenase-catalyzed reactions. While these metalloenzymes require trace metals as cofactors in their catalytic sites, these metals may be toxic at elevated concentrations. In this study, the effects of transition metals on 1,4-dioxane biodegradation by Pseudonocardia dioxanivorans CB1190, a monooxygenase-expressing bacterium, were investigated. Dose-dependent inhibition of 1,4-dioxane biodegradation by Cd(II), Cu(II), and Ni(II) was observed, whereas Zn(II) had no measurable effect on biodegradation rates. 1,4-Dioxane biodegradation in cultures exposed to 2 mg/L Cu(II) was restored in the presence of 0.005, 0.05, and 0.5 mM alginin, 0.05, and 0.5 mM cysteine, and 0.005 mM tannin. These results indicated that specific ligands bind with transition metals and alleviate bacterial toxicity. In parallel experiments, tannin and cysteine inhibited 1,4-dioxane biodegradation, but alginin, BSA, and SRNOM did not affect the biodegradation rates. Thus, monooxygenase-catalyzed biodegradation rates are subject to interactions among transition metals and natural organic ligands in the environment. Mechanistic insights and quantitative data obtained in this study will be useful for designing bioremediation strategies at sites simultaneously contaminated with metals and organic pollutants.