The current study investigated the performance of a moving bed biofilm reactor (MBBR), when adding 3D-printed biocarriers fabricated with 13X and bentonite (MBBR 3D), when using K1 commercial biocarriers (MBBR K1) and when not adding biocarriers at all (control MBBR). For the evaluation of the MBBR efficiency, various physicochemical parameters were measured, while 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 processes were improved in MBBR 3D 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 in the K1 biocarriers. Moreover, in the K1 biocarriers the mass of the biofilm varied in relation to time, since it could not be protected inside the holes, something that did not happen with the 3D-printed biocarriers. Finally, it was found, mostly in MBBR 3D and less in MBBR K1, that the growth of nitrifying bacteria and heterotrophs inside the units increased the biomass production in the form of soluble microbial products, which in turn favored the adhesion of biomass on the surface of biocarriers.