Enhanced in situ biodenitrification (EIB) is a feasible technology to clean nitrate-polluted groundwater and reach drinking water standards. Aimed at enabling a better monitoring and management of the technology at the field scale, we developed a two-dimensional reactive transport model (RTM) of a cross section (26.5 × 4 m) of a fractured aquifer composed of marls involving both biogeochemical processes and associated isotope fractionation. The RTM was based on the upscaling of a previously developed batch-scale model and on a flow model that was constructed and calibrated on in situ pumping and tracer tests. The RTM was validated using the experimental data provided by Vidal-Gavilan et al. (2013). The model considers several processes including (i) exogenous and endogenous microbial nitrate and sulfate respiration coupled to ethanol oxidation and linked to microbial growth and decay, and (ii) geochemical interactions (dissolution/precipitation of calcite), and (iii) isotopic fractionation of the reaction network (15N–NO3, 18O–NO3, 13C–DIC, 13C–ethanol, 13C–biomass, and 13C–calcite). Most of the calibrated microbiological parameter values at field scale did not change more than one order of magnitude from those obtained at batch scale, which indicates that parameters determined at the batch scale can be used as initial estimates to reproduce field observations provided that groundwater flow is well known. In contrast, the calcite precipitation rate constant increased significantly (fifty times) with respect to batch scale. The incorporation of isotope fractionation into the model allowed to confirm the overall consistency of the model and to test the practical usefulness of assessing the efficiency of EIB through the Rayleigh equation approach. The large underestimation of the Rayleigh equation of the extent of EIB (from 10 to 50%) was caused by the high value of hydrodynamic dispersion observed in this fractured aquifer together with the high reaction rates.