The reduction of biological excess sludge production using ozone is a well-known technology and is applied in several full-scale plants around the world. Nevertheless, optimisation of the process is not yet adequately documented in the literature. Operational parameters are usually chosen by assuming a direct proportionality between ozone dose and excess sludge reduction. This paper investigates the role of ozone concentration on process efficiency and demonstrates the (non-linear) inverse relationship between ozone dose and specific particulate chemical oxygen demand solubilisation in plug-flow contact reactors. The influence of total suspended solids concentration is also studied and described. No short-term lethal effects on heterotrophic biomass have been observed.