Monooxygenation is an important route of nitroaromatic compound (NAC) biodegradation and it is widely found for cometabolic transformations of NACs and other aromatic pollutants. We investigated the C and N isotope fractionation of nitrophenol monooxygenation to complement the characterization of NAC (bio)degradation pathways by compound-specific isotope analysis (CSIA). Because of the large diversity of enzymes catalyzing monooxygenations, we studied the combined C and N isotope fractionation and the corresponding ¹³C- and ¹⁵N-apparent kinetic isotope effects (AKIEs) of four nitrophenol-biodegrading microorganisms (Bacillus spharericus JS905, Pseudomonas sp. 1A, Arthrobacter sp. JS443, Pseudomonas putida B2) in the pH range 6.1–8.6 with resting cells and crude cell extracts. While the extent of C and N isotope fractionation and the AKIE-values varied considerably for the different organisms, the correlated C and N isotope signatures (δ¹⁵N vs δ¹³C) revealed trends, indicative of two distinct monooxygenation pathways involving hydroxy-1,4-benzoquinone or 1,2- and 1,4-benzoquinone intermediates, respectively. The distinction was possible based on larger secondary ¹⁵N-AKIEs associated with the benzoquinone pathway. Isotope fractionation was neither masked substantially by nitrophenol speciation nor transport across cell membranes. Only when 4-nitrophenol was biodegraded by Pseudomonas sp. 1A did isotope fractionation become negligible, presumably due to rate-limiting substrate binding steps pertinent to the catalytic cycle of flavin-dependent monooxygenases.