Aerobic biodegradation of individual mononitrophenols (4-, 3- and 2-NPs) and their mixture in simulated wastewater was investigated in a packed-bed bench scale bioreactor continuously operated in a flow mode, with a mixed microbial culture adsorbed on expanded slate. Under a low, suboptimal hydraulic retention time (HRT) of 30 min the reactor removed more than 3 g.L(-1).day(-1) of the NP mixture while maintaining a > 85-90% removal efficiency (RE). Under higher HRT values, starting at 45 min, more than 2 g.L(-1).day(-1) of the NP mixture were removed with an RE > 98%. Significant substrate interactions were observed; the addition of other NPs caused the saturation of 2-NP catabolic capacity whereas the addition of 2-NP caused the de-saturation of the 4- and 3-NP catabolic capacity. 3- and 4-NPs appeared to be removed independently, i.e., by different enzyme systems. After ten months of operation, the biofilm composition was significantly altered to become predominantly bacterial. Only one originally inoculated strain remained indicating microbial contamination followed by a genetic material exchange.