This study evaluates the potential of bio-augmentation to improve the degradation of recalcitrant nonylphenol during the wastewater treatment in membrane bioreactors (MBR). One MBR containing activated sludge was bio-augmented using multistep inoculation with freeze dried Sphingomonas sp. strain TTNP3, whereas a second control reactor contained activated sludge solely. The C-14-labeled-nonylphenol isomer (4-[1-ethyl-1,3-dimethylpentyl]phenol) was applied as a single pulse. Bio-augmentation resulted in an immediate increase of dissolved radioactivity in the effluent in comparison to the control reactor (13% and 2% of initially applied radioactivity after 1 day, respectively). After 5 days of operation, the retentate of the bio-augmented reactor contained only 7% of the initial radioactivity in contrast to 50% in the control reactor. The radioactivity associated to the mixed liquor suspended solids, i.e., the suspension of biomass and other solids on the retentate side of the membrane, was mainly found as non-extractable residues that were increasingly formed during prolonged reactor operation, especially for the control MBR. HPLC-LSC and GC-MSn analyses revealed that the bio-augmented reactor produced more polar hydroquinone as main degradation intermediate, whereas the control reactor effluent contained a complex mixture of apolar compounds with shortened oxidized alkyl chains. Thus, the apparent differences in the behavior of nonylphenol between the reactors were due to the catabolism of nonylphenol conferred by bio-augmentation with Sphingomonas sp. strain TTNP3.