To examine the effects of the ozonation process (as an oxidation treatment for water and wastewater treatment applications) on microbial biofilm formation and biodegradability of organic compounds present in oil sands process-affected water (OSPW), biofilm reactors were operated continuously for 6 weeks. Two types of biofilm substrate materials: polyethylene (PE) and polyvinylchloride (PVC), and two types of OSPW—fresh and ozonated OSPWs—were tested. Endogenous microorganisms, in OSPW, quickly formed biofilms in the reactors. Without ozonation, the bioreactor (using endogenous microorganisms) removed 13.8% of the total acid-extractable organics (TAO) and 18.5% of the parent naphthenic acids (NAs) from fresh OSPW. The combined ozonation and biodegradation process removed 87.2% of the OSPW TAO and over 99% of the OSPW parent NAs. Further UPLC/HRMS analysis showed that NA biodegradability decreased as the NA cyclization number increased. Microbial biofilm formation was found to depend on the biofilm substrate type.