Abstract Deep-sea carbonate mounds can harbor a wide variety of heterotrophic and chemosynthetic microbial communities, providing biodiversity hotspots among the deep-sea benthic ecosystems. This study examined the bacterial and archaeal diversity and community structure in the water column and sediments associated to a recently described giant carbonate mound named Alpha Crucis Carbonate Ridge (ACCR), Southwestern Atlantic. Due to the acoustic evidence of gas chimneys from a previous study, we further evaluated the chemosynthetic primary production through in situ-simulated dark carbon fixation rates. Pelagic microbial communities varied significantly with depth, showing a high abundance of photosynthetic groups in surface waters and taxa related to nitrification in intermediate and deep waters. The benthic communities from the top of the ACCR were very similar along with the sediment depth, while those from the base of ACCR showed a clear stratification pattern, with members in the deep strata mainly related to anoxic and chemosynthetic ecosystems. Dark carbon fixation rates were of the same order of magnitude as those of deep-sea cold seeps and hydrothermal vents. Our study provides the first description of the ACCR microbiome and adds new information to help formulate and implement future conservation and management strategies for vulnerable marine ecosystems.