Industrial waste products may be a viable alternative to commercial external carbon sources. In this study, the properties of fusel oil (distillery waste product) were investigated in terms of temperature dependency of denitrification and acclimation period. Furthermore, results obtained during three different full-scale trials were used to verify similarities with and differences from the results obtained under laboratory-scale conditions. Batch experiments with non-acclimated biomass revealed a very strong temperature dependency of the denitrification process (θ = 1.15, R2 = 0.92) in the range of examined process temperatures (13–22 °C). Fusel oil had minor (or no) effects on the behaviour of NO2-N and PO4-P. Significantly lower nitrate utilization rates were observed during acclimation to fusel oil in the full-scale bioreactors compared to a bench-scale reactor. This may primarily be attributed to lower doses of fusel oil, lower process temperatures and more complicated process configurations (resulting in non-optimal use of fusel oil for denitrification). Results obtained from both laboratory-scale experiments and full-scale trials suggested that an acclimation period of a few weeks would be required to reach the maximum denitrification capability of process biomass, even though positive effects of dosing can be observed almost immediately.