This study investigates the potential of hydrodynamically disintegrated excess activated sludge when used as a supplementary carbon source for denitrification. Two objectives constituted this study: (i) to analyse the denitrification rate by using excess sludge subjected to hydrodynamic disintegration (HD), performed at different energy densities, as an organic carbon source, and (ii) to analyse the impact of hydrolysis of disintegrated sludge on the denitrification rate. Nitrate reduction tests were conducted to assess the denitrification rate for the following sources of organic carbon: thickened excess sludge disintegrated at three levels of energy density (70, 140 and 210 kJ/L), acetic acid solution and municipal wastewater after mechanical treatment. It was found that the HD of excess sludge conducted at different levels of energy density led to dissolved organic compounds characterised by various properties as donors of H+ in the denitrification process. The susceptibility of disintegrated sludge to anaerobic hydrolysis decreased along with the increasing energy density. The obtained organic carbon contributed to a lower increase in the denitrification rate in comparison to that when disintegrated sludge not subjected to hydrolysis was applied.