Degradations of nitrotoluenes, individually and in a mixture, were carried out in batch and continuous aerobic reactors by a defined mixed microbial culture. The degradation rates and efficiencies of the isomers were evaluated in batch and continuous reactors. The results demonstrated that all the three-mononitrotoluene isomers were degraded simultaneously and completely in presence of excess oxygen. The nitro group position on the benzene ring influenced the degradation rates of the individual MNT isomers in batch systems. In the continuous biofilm reactor with a sufficient biocatalyst quantity, quality and excessive oxygen supply rate, the degradation rates of the mononitrotoluenes were almost identical as long as the compounds were present individually and their loading did not exceed the capacity of the catabolic master reaction. The microbial composition of the biofilm changed qualitatively and quantitatively during long-term continuous operation under aerobic non-aseptic conditions. This complex investigation resulted in data that can be applied for the scale-up procedure for field experiments.