This study evaluates the effect of biofilm thickness on the nitrifying activity in moving bed biofilm reactors (MBBRs) in a controlled environment. In-depth understanding of biofilm properties in MBBRs and their effect on the overall treatment efficiency is the key to optimizing process stability and efficiency. However, evaluating biofilm properties in continuously operated MBBRs can be extremely challenging. This study uses a carrier design which enables comparison of four different biofilm thicknesses, in otherwise equally operated lab-scale MBBRs. The results show that within the studied range (200-500 µm) and specific operation conditions, biofilm thickness alone had no significant effect on the overall ammonium removal. The nitrate production, however, decreased with a decreasing biofilm thickness, and the ratio between nitrite and ammonia-oxidizing activity decreased both with increasing load and decreasing oxygen concentration for all thicknesses. The suggestion that nitratation is disfavoured in thin biofilms is an interesting contribution to the current research being performed on nitrite-oxidizing bacteria inhibition for deammonification applications. By indicating that different groups of bacteria respond differently to biofilm thickness, this study accentuates the importance of further evaluation of these complex systems.