Pre-ozonation is often used to enhance the biodegradability of recalcitrant compounds prior to biological treatment of wastewater. A usual shortcoming of such an approach is wasting ozone on other compounds that are already biodegradable. This research followed a groundbreaking approach of degrading a recalcitrant substance with ozone during biological treatment. Two parallel bench-top activated sludge processes were fed a synthetic wastewater containing typical biodegradable substances and also methylene blue at 5 mg/L. Ozone was applied continuously and directly into one of the activated sludge units at 17 mg/L based on inflow rate. The methylene blue was removed by 95% in the ozonated process compared with just 40% removal in the non-ozonated control. The removal in the activated sludge without ozonation was demonstrated to be mainly due to biosorption. The ozone oxidation reaction by-products were analyzed using GC-MS on volatile substances collected in the headspace above ozonated samples of methylene blue and most found to be biodegradable. These by-products are expected to be degraded and assimilated in the same process unit together with the other biodegradables in the feed stream by the activated sludge process. The reaction rate with organic substances depleted the dissolved ozone at such a rate that the inactivation of the treatment bacteria (and protozoa) was minimal, mostly affecting the filamentous bacteria. A concern that ozone, as a powerful disinfectant, could inhibit or kill the beneficial bacteria in the activated sludge process was proven to be incorrect.