This study aims to investigate the biodegradation of 4,5-dichloro-2-(n-octyl)-3[2H]-isothiazolone (DCOIT) by a brown-rot fungus Gloeophyllum trabeum as well as the involved mechanism. In the present study, the retentions of DCOIT in treated Masson pine (Pinus massoniana) (MP) chips were determined periodically after incubation with G. trabeum. Then a Fenton-like reaction, known as the chelator-mediated Fenton (CMF) chemistry was used to degrade DCOIT that mimics the degradation pathway of DCOIT by typical brown-rot fungi, and the degradation intermediates were further analyzed. The results demonstrated that DCOIT was rapidly depleted in the early stages of incubation by G. trabeum. The CMF treatment was shown to oxidatively decompose DCOIT by producing reactive oxygen species. This evidence suggests that the CMF chemistry employed by brown-rot fungi contributes to the rapid depletion of DCOIT during G. trabeum exposure, although this does not rule out other possible mechanisms for the biodegradation of DCOIT. The new findings give new insights into the mechanism for the biodegradation of organic biocides, and potentially provide an efficient approach for the removal of organic pollutants in the contaminated water.