Naturally bioluminescent fungi, Armillaria mellea and Mycena citricolor, were used to develop a novel, bioluminescence-based bioassay for toxicity testing. Bioassays were carried out to assess the toxicity of 3,5-dichlorophenol (3,5-DCP), pentachlorophenol (PCP), copper and zinc. The results suggested that 60 min was a suitable exposure time for the bioassay. Light reduction was observed in response to 3,5-DCP, PCP and Cu for both A. mellea and M. citricolor, but to Zn only for A. mellea. Armillaria mellea was significantly less sensitive to 3,5-DCP and PCP than M. citricolor. The EC50 values for A. mellea and M. citricolor were similar to EC50 values for 3,5-DCP, PCP and Cu (but not Zn) of bioluminescence-based bacterial biosensors. They were also similar to EC50 values for Cu and Zn of a bioluminescence-based yeast biosensor. The results highlighted the importance of using both prokaryotic and eukaryotic biosensors. The novel bioassay provides a rapid and sensitive method to assess bioavailability of pollutants as well as a method to determine their toxicity to filamentous fungi. It also expands the range of organisms that can be used for bioluminescence-based toxicity testing by complementing existing biosensors.