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Microbial communities associated with marine sponges carry out nutrient transformations essential for the benthic-pelagic coupling however, knowledge about their composition and function is still sparse. We evaluated the richness and diversity of prokaryotic assemblages associated with three high-microbial abundance (HMA) and three low-microbial abundance (LMA) sympatric Mediterranean sponges to address their stability and uniqueness. Moreover, to examine functionality and because an imbalance between nitrogen ingestion and excretion has been observed on some of these species, we sequenced nitrogenase genes (nifH) and measured N2 fixation. The prokaryotic communities in the two sponge types did not differ in terms of richness, but highest diversity was found in HMA sponges. Moreover, the discrete composition relative to surrounding seawater indicated that horizontal and vertical transmission affect the microbiomes associated with both sponge categories. nifH genes were found in all LMA and sporadically in one HMA species, and about half of these sequences were common between the different sponge species and also, they were found in the surrounding water suggesting horizontal transmission. 15N2–enriched incubations showed that N2 fixation was measurable in the water but not associated with the sponges. Also, the analysis of the isotopic ratio of 15N to 14N of sponge tissue indicated that N2 fixation was not an important source of nitrogen in these Mediterranean sponges. Overall, our results suggest that compositional and functional features differ between prokaryotic communities associated with HMA and LMA sponges, which may affect sponge ecology.