In this study, two moving-bed biofilm reactors (MBBR₁ and MBBR₂) filled with different size of carrier media (Kaldnes K₁ and Kaldnes K₁ micro, respectively) were subjected to soluble (sugar and sodium acetate (Ac)) substrate and mixture of soluble and particulate (particulate potato starch (PS)) substrate in a very high organic loading rate (12 kgCOD/m³·d) at different temperatures (26 and 15°C, in MBBR₁ and MBBR₂, respectively). The effects of carrier type and substrate on biofilm structure and reactor performance have been studied. Starch was removed by adsorption at the biofilm surface and hydrolyzed which caused substrate gradient in MBBR₁, however, hydrolyzed uniformly within biofilm in MBBR₂. The biofilm of MBBR₁ was irregular due to filamentous structure growth due to the substrate gradient, while, it was regular in MBBR₂ due to uniform distribution of substrate. The performance of both MBBRs in ammonium, COD and TN removal decreased significantly when the amount of small particles in the reactor increased owing to feeding by starch, which led to biomass density decline. The type of media affected the quantity and distribution of attached biomass, which in turn influenced the activity of specific microbial functional groups in the biofilm. The biofilm in MBBR₂ was thicker and consequently nitrogen removal by denitrification was much higher. The lower temperature did not affect negatively the reactor performance in MBBR₂.