A heterogeneous respirometer (HR) was coupled for the first time to a microelectrode monitoring system specifically designed for dissolved oxygen (DO) measuring within the biofilm. Monitoring of the oxygen concentration in the gas and liquid phases was complemented with pioneer monitoring of DO performed simultaneously and continuously at multiple biofilm depths in a linear array of eleven gold-disk electrodes of 50 µm-diameter. A set of respirometric tests performed at neutral pH and with initial gas phase concentrations of H2S ranging from 135 to 6720 ppmv were used to assess sulfide-oxidizing activity of a biofilm grown on 15.9 mm plastic Pall rings withdrawn from a biogas desulfurizing biotrickling filter. A mechanistic model for the description of multi-step sulfide oxidation within a biotrickling filter was improved considering heterogeneous biomass concentration and biomass activity distribution along the biofilm depth. A comprehensive description of physical, chemical and biological phenomena occurring throughout gas, liquid and biofilm phases resulted in an accurate prediction of system behavior. Model calibration using experimental data estimated a biomass concentration from 320.5 to 443.0 g VSS·L−1 as well as a decrease in the fraction of active biomass of 0.5, over the 600 µm thick biofilm. Model simulations accurately reproduced experimental respirometric profiles (NRMSE <10%), demonstrating that coupling HR and microelectrodes improved model predictions in comparison to sole gas or liquid phase measurements, thus contributing to a deeper knowledge of biofilms performance in trickled bed biological systems.