Iron-containing liquid cultures of the brown-rot basidiomycete Gloeophyllum striatum degraded 2-fluorophenol. Two simultaneously appearing degradation products, 3-fluorocatechol and catechol, were identified by gas chromatography and mass spectrometry (GC-MS). Concomitantly, fluoride was produced at approximately 50% of the amount that theoretically could be achieved upon complete dehalogenation. Defluorination was strongly inhibited in the presence of either the hydroxyl radical scavenger mannitol or superoxide dismutase, as well as in the absence of iron. The addition of the natural iron chelator oxalate caused a clear but less extensive inhibition, whereas supplementation with the artificial iron chelator nitrilotriacetic acid increased fluoride production. Extracellular 2-fluorophenol degradation was evidenced by defluorination, observed upon addition of 2-fluorophenol to cell-free culture supernatants derived from iron-containing fungal cultures. Ultrafiltered culture supernatants oxidized methanol to formaldehyde, known as a product of the reaction of methanol with hydroxyl radical. In addition, G. striatum was found to produce metabolites extractable with ethyl acetate that are capable of reducing Fe3+. GC-MS analysis of such extracts revealed the presence of several compounds. The mass spectrum of a prominent peak matched those previously reported for 2,5-dimethoxyhydroquinone and 4,5-dimethoxycatechol, fungal metabolites implicated to drive hydroxyl radical production in Gloeophyllum. Taken together, these findings further support an extracellular Fenton-type mechanism operative during halophenol degradation by G. striatum.