A large variety of microbial parameter values for nitrifying microorganisms has been reported in literature and was revised in this study. Part of the variety was attributed to the variety of analysis methods applied; it also reflects the large biodiversity in nitrifying systems. This diversity is mostly neglected in conventional nitrifying biofilm models. In this contribution, a one-dimensional, multispecies nitrifying biofilm model was set up, taking into account the large variety of the maximum growth rate, the substrate affinity and the yield of nitrifiers reported in literature. Microbial diversity was implemented in the model by considering 60 species of ammonia-oxidizing bacteria (AOB) and 60 species of nitrite-oxidizing bacteria (NOB). A steady-state analysis showed that operational conditions such as the nitrogen loading rate and the bulk liquid oxygen concentration influence both the macroscopic output as well as the microbial composition of the biofilm through the prevailing concentration of substrates throughout the biofilm. Considering two limiting resources (nitrogen and oxygen), the coexistence of two species of the same functional guild (AOB or NOB) was possible at steady state. Their spatial distribution in the biofilm could be explained using the r- and K-selection theory. Biotechnol. Bioeng. 2015;112: 2550-2561. (c) 2015 Wiley Periodicals, Inc.