Naphthenic acids (NAs) are a complex group of alkyl-substituted acyclic, monocyclic and polycyclic carboxylic acids present in crude oil, oil sands process water and tailings ponds, as well as in refinery wastewater. Bioassays were performed to investigate the biotransformation potential and inhibitory effect of a commercial NA mixture to nitrification, denitrification and fermentation/methanogenesis using mixed cultures not previously exposed to NAs. NAs inhibited nitrification in a mixed aerobic heterotrophic/nitrifying culture at concentrations as low as 80 mg NA/L, whereas, an enriched nitrifying culture was only affected at 400 mg NA/L. The lower nitrification inhibition in the latter assay is attributed to the higher population size of nitrosofying and nitrifying bacteria compared to the mixed heterotrophic/nitrifying culture. The NA mixture was not inhibitory to denitrifiers up to 400 mg/L. At higher NA concentrations, cell lysis was pronounced and lysis products were the main source of degradable carbon driving denitrification in culture series prepared without an external carbon source. In the presence of a degradable external carbon source, no difference was observed in nitrate reduction rates or nitrogen gas production at all NA concentrations tested. Methanogenesis was completely inhibited at NA concentrations equal to or higher than 200 mg/L. Methanogenic culture series amended with 80 mg NA/L were transiently inhibited and methane production in culture series prepared with NAs and an external carbon source or NAs only recovered in 136 and 41 days, respectively. Accumulation of volatile fatty acids was observed at inhibitory NA concentrations; however, carbon dioxide production was not affected by NAs, indicating that fermentation and acidogenesis were not affected by NAs. NAs were not degraded under nitrate-reducing or fermentative/methanogenic conditions used in the present study, regardless of the presence or not of another, degradable carbon/energy source.