Pollutant interactions during the aerobic biodegradation of phenolic mixtures with either 2-nitrophenol (2-NP) or 3-nitrophenol (3-NP) by a NP-adapted microbial consortium in simulated wastewater were studied in a packed-bed bench scale bioreactor continuously operated in a flow mode. Phenol/2-NP and phenol/3-NP mixtures with varied phenol/nitrophenol ratios were shown to exhibit different biodegradability patterns. The presence of 2-NP led to a much lower overall elimination capacity and lower process stability in comparison to mixtures with 3-NP. In contrast to the expected greater degradation of a more biodegradable substrate in mixtures, phenol was degraded with a lower efficiency at higher phenol concentrations than NPs, although this difference became less pronounced with the gradual biofilm adaptation to phenol. This unusual substrate interaction, which appears to be common in the biotreatment of substituted phenol mixtures, was explained by prior biofilm adaptation to less degradable substrates, NPs. The biofilm composition was significantly altered during the long-term reactor operation. Although eukaryotes were not present in the inoculum, four fungal species were isolated from the biofilm after 1.5 years of operation. Of the initially present strains, only Chryseobacterium sp. and several Pseudomonas species persisted till the end of operation.