Biofilm models are valuable tools for process engineers despite several uncertainties including the dynamics and rate of biofilm detachment, concentration gradients external to the biofilm surface, and undefined biofilm reactor model calibration protocol. The present investigation serves to (1) evaluate two ad hoc model calibration procedures, (2) systematically evaluate critical biofilm model assumptions and components, and (3) conduct a sensitivity analysis with the aim of identifying parameter subsets for biofilm reactor model calibration. AQUASIM was used to describe submerged-completely mixed combined carbon oxidation and nitrification IFAS and MBBR systems, and tertiary nitrification and denitrification MBBRs. The magnitude of model output dependency on uncertainties in model parameters was determined by means of a sensitivity analysis. The following conclusions were drawn from this work. Ad-hoc expert-based trial and error calibration may have limited applicability as resulting parameters are often not sufficiently general to predict system performance during other periods, even for the same wastewater treatment plant. At both temperatures simulated (T = 20°C and 12°C), each system modeled was sensitive to changes in the biofilm parameter mass transfer boundary layer thickness (L L ). Sensitivity of model predictions to kinetic and biofilm parameters is strongly influenced by temperature. A cold temperature local sensitivity analysis provided evidence that biofilm models are sensitive to changes in mass transfer boundary layer thickness (L L ), diffusivity coefficient (D), maximum growth rate (µ), and affinity constant (K) for the ratelimiting substrate. No simple recommendations for the more appropriate model calibration methodology can be suggested as sensitivity of model predictions significantly depends on environmental conditions (e.g., temperature) and treatment objectives (e.g., nitrification, denitrification).