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The main aim of the study was to evaluate hydrogen and carbon isotope fractionation during biodegradation of benzene as a possible tool to trace the process in contaminated environments. Aerobic biodegradation of benzene by two bacterial isolates, Acinetobacter sp. and Burkholderia sp., was accompanied by significant hydrogen and carbon isotope fractionation with hydrogen isotope enrichment factors of −12.8 ± 0.7‰ and −11.2 ± 1.8‰, respectively, and average carbon isotope enrichment factors of −1.46 ± 0.06‰ and −3.53 ± 0.26‰, respectively. Inorganic carbon produced by Acinetobacter sp. was depleted in ¹³C by 3.6−6.2‰ as compared to the initial δ¹³C of benzene, while the produced biomass was enriched in ¹³C by 3.8‰. The secondary aim was to determine isotope ratios of benzenes from different manufacturers with regard to the use of isotopes for source differentiation. While two of the four …