This study highlights the effects of interaction between microbial, macro- and meiofauna on NO3−-N and DOC reduction in macroporous stream sediment. The tested hypotheses are: the transformation of nutrients and dissolved organic matter (1) is influenced by the presence of invertebrates, (2) is more effective when the diversity of the vertical benthic community increases. These hypotheses were tested using microcosms reproducing a portion of a river bed water-sediment interface that was colonized with different levels of invertebrate biodiversity. Experimental treatments were abiotic sediment (AS); sediment and biofilm (SB); sediment, biofilm and meiofauna (SBM); and sediment, biofilm, meiofauna and macrofauna community assemblage, which corresponds to the total benthic community of a river bed (SBMM). Reduction rates of nitrates (NO3−-N) and dissolved organic carbon (DOC) in the microcosms were measured and considered as a function of the different levels of biodiversity. Nutrient reduction rates were monitored by their decrease from the aqueous phase. Nitrate reduction rates increased significantly with increasing the vertical biodiversity level. After 56 days of biofilm development, NO3−-N reduction rates ranged from 3.76 ± 0.35 in SB treatment to 8.92 ± 0.69 mg N d−1 kg−1sediment Fresh Weight (sed FW) in the treatment with the maximum biodiversity (SBMM). Denitrification rates increased by a factor of 6 in presence of meiofauna and macrofauna compared to that measured in sediment without invertebrates. DOC reduction rates also varied significantly with biodiversity levels but in a lesser extent than nitrate reduction rates (41.89 ± 2.24 mg C d−1 kg−1sed FW with biofilm alone (SB) to 51.00 ± 1.39 mg C d−1 kg−1sed FW with the addition of meiofauna community) (SBM).