Carbon sources such as methanol and glycerol are used for enhancing denitrification at wastewater treatment plants, which are required to meet increasingly stringent effluent nitrogen limits. Consequently, dosing strategies for these compounds could benefit from the development and application of molecular activity biomarkers to infer and distinguish between methanol- or glycerol-based denitrification in activated sludge. In this study, the applicability of genes coding for methanol dehydrogenase (mdh2 and mxaF) and glycerol dehydrogenase (dhaD) as potential biomarkers of denitrification activity using these specific substrates was explored and confirmed using a two-pronged approach. First, during short-term spikes of activated sludge biomass with glycerol, the ability of dhaD mRNA concentrations to closely track nitrate depletion profiles was demonstrated. Second, a high-degree of correlation of the mRNA concentrations of mdh2, mxaF and dhaD with methanol- and glycerol-based denitrification kinetics during long-term bioreactor operation using these substrates was also shown. Based on these results, expression of mdh2, mxaF and dhaD genes are promising biomarkers of in situ denitrification activity on methanol and glycerol, respectively, in mixed-culture engineered wastewater treatment processes.