Flumequine, a fluoroquinolone antibiotic, is applied preferably in veterinary medicine, for stock breeding and treatment of aquacultures. Formation of drug resistance is a matter of general concern when antibiotics such as flumquine occur in the environment. Thus, biodegradation of flumequine in solution was investigated using five different ligninolytic fungi. Irpex lacteus, Dichomitus squalens, and Trametes versicolor proved most efficient and transformed more than 90% of flumequine within 6 or even 3 days. Panus tigrinus and Pleurotus ostreatus required up to 14 days to remove >90% of flumequine. Analyses of the metabolites by liquid chromatography–mass spectrometry suggest different transformation pathways for the different fungal strains. Structure proposals were elaborated for 8 metabolites. 7-Hydroxy-flumequine and flumequine ethyl ester were identified as common metabolites produced by all ligninolytic fungi. The largest variety of metabolites was formed by D. squalens. Residual antibacterial activity of the metabolite mixtures was tested using gram-positive and gram-negative bacteria. While for the less efficient P. tigrinus and P. ostreatus cultures the antibacterial activities corresponded to the residual concentrations of flumequine, a remarkable antibacterial activity remained in the D. squalens cultures although flumequine was transformed to more than 90%. Obviously, antibacterially active transformation products were formed by this fungal strain.