Seasonally ephemeral streams are common globally, and are inhabited by organisms that survive long dry periods, then capitalize on resources mobilized as flow resumes. In the McMurdo Dry Valleys of Antarctica, ephemeral streams contain diverse food webs supported by microbial autotrophy. Here, benthic microbial mats dominated by nitrogen (N-)fixing Nostoc spp., or “black” mats, provide N for autotrophy in neighboring Oscillatoriacean dominated, or “orange” mats, and for heterotrophy and N-cycling activity in hyporheic sediments. This study sought to understand the abundance, diversity, and functional potential of these microbial communities: We predicted that bacterial diversity, abundance, and N-cycling potential would be higher in black than orange mats and underlying sediments. Samples were collected in December 2023 (early austral summer, before flow started) and analyzed for bacterial 16S rRNA, amoA, and nosZ gene copies as total and functional population proxies, and 16S and 18S rRNA gene sequence libraries to assess microbial diversity and composition. Gene abundances were similar in black and orange mats (P>0.08), but tended to be higher in sediments underlying black mats, due to higher DNA concentrations (P<0.05), suggesting greater energy flow from black mats to underlying sediments. While diversity was similar in and under black and orange mats (P>0.4), bacterial sequence variant composition was strongly structured by habitat type (mat vs. sediment) and pigment color (together >35% of variance, P<0.001). Specifically, hyporheic sediments contained more bacterial taxa in common with orange mats, suggesting stronger dispersal connectivity from orange mats. In contrast, eukaryotic communities were weakly structured at the sequence variant level, but showed clear differences in autotrophic and heterotrophic food web components between mat and sediment habitats. Overall, Nostoc-dominated benthic mats support more energy input, while Oscillatoriacean-dominated mats provide more diversity, to underlying hyporheic sediments in these ephemeral streams, and together support a mosaic of diverse microbial food webs.