A cell-free culture broth of Phanerochaete chrysosporium immobilized in polyurethane foam has been evaluated for the biodegradation of chlorophenols. Lignin peroxidase, manganese peroxidase, and oxalate concentrations in cell-free culture broth were measured and compared to find the optimum combination of secondary metabolites for the highest biodegradation of chlorophenols. The isozyme distributions and their expression levels were significantly different and changed with increases in the culture time. The oxalate concentration was also found to vary, depending on culture time. Cell-free broth containing an optimal combination of secondary metabolites showed the greatest biodegradation of 2,4,5-chlorophenol in the presence of veratryl alcohol and H2O2. Phenols with greater numbers of chlorines were degraded more efficiently by this cell-free culture broth according to the results of biodegradation experiments for five chlorophenols, including 2-, 4-, 2,4-, 2,4,5-, and pentachlorophenol, as well as phenol. This degradation efficiency correlated well with remaining lignin peroxidase activity during degradation. Cell-free culture broths may readily be used for biodegradation of highly recalcitrant chemicals since the system would not be affected by the toxicity of the chemicals nor would the adsorption characteristics of the cells be of concern.