Supernatants of mycelial cultures of seven basidiomycetous fungi indigenous to agricultural sites were evaluated for metabolites generated from the veterinary fluoroquinolone enrofloxacin (EFL) by employing high-performance liquid chromatography/high-resolution electrospray ionization mass spectrometry. From exact masses, molecular formulae were derived, and the most probable chemical structures were deduced. Patterns of major metabolites were surprisingly similar but differed greatly from that provided by Gloeophyllum striatum due to the absence of monohydroxylated EFL congeners and a greater variety of metabolites with a modified piperazine moiety. The structures of three metabolites were elucidated by 1H-nuclear magnetic resonance spectroscopy. Of 61 compounds detected, 48 were new, while 13 were known from a pattern of 87 EFL metabolites identified for G. striatum. Ethylpiperazine moieties carrying oxido, hydroxy, oxo, and acetoxy groups, or showing partial degradation, were linked to the unmodified, oxidatively decarboxylated, or multiply hydroxylated core of EFL and to isatin- and anthranilic acid-type EFL congeners. Cleavage of the fluoro-aromatic bond was observed for two, 14CO2 formation for six species. Metabolites with a hydroxylated aromatic part implied subsequent ring cleavage to be brought about by the formation of potentially four oxidizable ortho-aminophenol- and one catechol-type intermediates. EFL degradation appears to be a common activity among basidiomycetes.