Abstract A rotating tube bioreactor (RTB) was developed to examine chemical degradation by fungal biofilms under controlled laboratory conditions. Each RTB consists of a sealed tube containing mycelia growing on a piece of steel mesh on the inside wall of the tube. Tubes are mounted horizontally in a tube rotator and slowly rotated to expose the fungal biofilm to a wetting-aeration cycle typical of fixed film reactors such as trickling filters and rotating biological contactors. The extent of pentachlorophenol (PCP) dehalogenation by three different species of fungal biofilms was evaluated by measuring concentrations of PCP (using HPLC) and the total concentrations of organic and inorganic halides (using a TOX analyzer). The three species of fungi removed PCP from solution at similar rates, but they differed in ability to completely dehalogenate PCP. Nitrogen-deficient growth media only slightly increased the extent of PCP mineralization. All species of fungi (grown on nitrogen-sufficient media) removed PCP to <0.1 mg l −1 (>99% removal) within 1 d. Within 8 d, the greatest percent of dehalogenation, measured by the appearance of chloride ion in solution, was achieved by Trametes versicolor (62%), followed by Phanerochaete chrysosporium (38%) and Inonotus dryophilus (21%). These removals based on chloride ion concentrations are comparable to claims by others of complete mineralization for recoveries of 10–50% of 14CO 2 from 14C-labeled PCP in static flask cultures.