Pathways of N2 production and the influence of oxygen, nitrate/nitrite, and sulfide on the activity of denitrifying bacteria were studied in the chemocline of the shallow, brackish Mariager Fjord, by means of incubation experiments with 15N-labeled compounds. Denitrification was an important process, with potential rates up to 18.6 ± 0.12 µM N2 d− 1. The potential for denitrification increased with water depth across the chemocline towards higher hydrogen sulfide concentrations, which indicates the utilization of sulfide as an electron donor for denitrification. The anammox process was not detected, and therefore denitrification is the dominant sink for bioavailable nitrogen in Mariager Fjord. The absence of anammox may be explained by an inhibitory effect of low sulfide levels and/or an unstable environment caused by chemocline perturbations that generate unfavorable conditions for slow-growing anammox bacteria. Nitrate reduction to nitrite and denitrification (nitrite reduction to N2O and, mainly, N2) fueled by sulfide oxidation was evident from the direct dependence of rates on endogenous sulfide concentrations, with first-order rate constants of 1.90 ± 0.31 and of 0.93 ± 0.11 d− 1 for the two processes, respectively, and from the correlation of N2 production with sulfide consumption in anoxic incubations. Elemental sulfur (S0) was the immediate product of sulfide oxidation with both nitrate and nitrite. The nitrate and nitrite dependence of the processes followed Michaelis–Menten kinetics with higher apparent Km and Vmax for nitrate reduction to nitrite than for denitrification of nitrite, leading to an accumulation of nitrite at nitrate concentrations ≥ 5 µM. The apparent Km of nitrate consumption during denitrification was 2.9 ± 0.1 µM, which is close to or even higher than the range of nitrate concentrations encountered by denitrifying bacteria in the chemocline, and nitrate is therefore continuously a limiting factor for chemolithotrophic denitrification in Mariager Fjord. In addition to substrate availability, oxygen is an important controlling factor for denitrification and inhibited the process at concentrations of 8–15 µM. The experimentally-derived kinetics were validated for the conditions in situ in a 1-D reaction transport model, which reproduced the nitrate distribution at the chemocline well, suggesting that our incubation results provide a good quantitative description of the regulation of denitrification by nitrate and sulfide in situ. Our study demonstrates that the chemocline is an important site for removal of bioavailable nitrogen by chemolithotrophic denitrification in Mariager Fjord.