Syntrophic mineralisation of benzene, as recently proposed for a sulphate-reducing enrichment culture, was tested in product inhibition experiments with acetate and hydrogen, both putative intermediates of anaerobic benzene fermentation. Using [13C6]-benzene enabled tracking the inhibition of benzene mineralisation sensitively by analysis of 13CO2. In noninhibited cultures, hydrogen was detected at partial pressures of 2.4 × 10−6± 1.5 × 10−6 atm. Acetate was detected at concentrations of 17 ± 2 μM. Spiking with 0.1 atm hydrogen produced a transient inhibitory effect on 13CO2 formation. In cultures spiked with higher amounts of hydrogen, benzene mineralisation did not restart after hydrogen consumption, possibly due to the toxic effects of the sulphide produced. An inhibitory effect was also observed when acetate was added to the cultures (0.3, 3.5 and 30 mM). Benzene mineralisation resumed after acetate was degraded to concentrations found in noninhibited cultures, indicating that acetate is another key intermediate in anaerobic benzene mineralisation. Although benzene mineralisation by a single sulphate reducer cannot be ruled out, our results strongly point to an involvement of syntrophic interactions in the process. Thermodynamic calculations revealed that, under in situ conditions, benzene fermentation to hydrogen and acetate yielded a free energy change of ΔG′=−83.1 ± 5.6 kJ mol−1. Benzene mineralisation ceased when ΔG′ values declined below −61.3 ± 5.3 kJ mol−1 in the presence of acetate, indicating that ATP-consuming reactions are involved in the pathway.