In the present work, a comparative evaluation of an MBBR unit performance was carried out for the following cases: when adding 3D-printed biocarriers fabricated with 13X and bentonite, when using K1 commercial biocarriers and when not adding biocarriers at all. For the evaluation of the MBBR efficiency, various physicochemical parameters were measured, while static light scattering and optical microscopy observations were additionally used. Finally, biofilm extracted from the biocarriers was evaluated. The findings suggest that there is an optimal biodegradation of the organic load in all MBBR units. The nitrification and denitrification process were improved at the 3D MBBR as compared to the control MBBR and MBBR K1. The dry mass of the biofilm in the 3D-printed biocarriers was two orders of magnitude larger than the one in the K1 biocarriers. What is more, in the K1 biocarriers the mass of the biofilm varied in relation to time, due to the fact that it could not be kept inside the holes, something that was not observed to happen with the 3D-printed biocarriers. Finally, it was observed, mostly in the 3D MBBR and less in the K1 MBBR, that the growth of nitrifying bacteria and heterotrophs inside the units increased the biomass production in the form of SMP, which in turn favored the adhesion of biomass on the surface of biocarriers.