This work deals with a comparative analysis of two alternative anaerobic digestion models proposed by Batstone et al. [D.J. Batstone, J. Keller, I. Angelidaki, S.V. Kalyuzhnyi, S.G. Pavlostathis, A. Rozzi, W.T.M. Sanders, H. Siegrist, V.A. Vavilin, Anaerobic Digestion Model No. 1 (ADM1) IWA Task Group for Mathematical Modelling of Anaerobic Digestion Processes, IWA Publishing, London, UK, 2002] and Angelidaki et al. [I. Angelidaki, L. Ellegaard, B.K. Ahring, A comprehensive model of anaerobic bioconversion of complex substrates to biogas, Biotechnol. Bioeng. 63 (5) (1999) 363–372], and their application to biofilm systems. Bioreactors are modeled as dynamic (gas–solid–liquid) three-phase systems. The experimental set-up consists of two mesophilic (36 ± 1 °C) lab-scale anaerobic fluidized bed reactors, which were loaded with sand as inert support for biofilm development. The experimental protocol is based on step-type disturbances applied on the inlet substrate concentration (glucose and acetate-based feeding) and on the feed flow rate considering the criterion of maximum efficiency. The predicted and measured responses of total and soluble chemical oxygen demand (COD), volatile fatty acid concentrations, biogas production rate and pH are investigated. Under the operating conditions evaluated in this investigation, the anaerobic digestion model proposed by Angelidaki et al. [I. Angelidaki, L. Ellegaard, B.K. Ahring, A comprehensive model of anaerobic bioconversion of complex substrates to biogas, Biotechnol. Bioeng. 63 (5) (1999) 363–372] ensures the best prediction. Parameters related to non-active biomass composition, disintegration and hydrolysis should be revised to achieve a good agreement between experimental and predicted values using the model proposed by Batstone et al. [D.J. Batstone, J. Keller, I. Angelidaki, S.V. Kalyuzhnyi, S.G. Pavlostathis, A. Rozzi, W.T.M. Sanders, H. Siegrist, V.A. Vavilin, Anaerobic Digestion Model No. 1 (ADM1) IWA Task Group for Mathematical Modelling of Anaerobic Digestion Processes, IWA Publishing, London, UK, 2002].