Biofilm reactors are an alternative technology to the conventional activated sludge process for treating wastewater. Understanding the microbial activity microdistribution in heterogeneous biofilms will enable exploration of microbial microdistribution, substrate transfer, and reaction processes inside biofilms. However, the microbial activity obtained from models and the reported measurement techniques are imprecise and complex, respectively. In this study, a convenient approach for estimating the relative microbial oxygen metabolic activity microdistribution inside biofilms is proposed. The oxygen-infusion processes within the biofilm produced by a rotating biological contactor (RBC) were measured by an oxygen microelectrode system. The measurement results showed that there were regular catastrophe points of dissolved oxygen (DO) concentration vs. time, and the DO concentration change rate increased notably at the catastrophe points. This catastrophe point phenomenon was observed for the first time and was analyzed using the three-dimensional diffusion-reaction model. Based on the analysis, it appears that the catastrophe point begins the biomass-limited stage. These points can be used to estimate the relative microbial oxygen metabolic activity microdistribution inside biofilms. This proposed method is convenient and less destructive than other methods. This approach enables the exploration of oxygen transfer-reaction micro-processes, dynamic OUR microdistribution, and microbial microdistribution inside biofilms.