The long-term dynamics of microbial communities across geographic, hydrographic, and biogeochemical gradients in the Arctic Ocean are largely unknown. To address this, we annually sampled polar, mixed, and Atlantic water masses of the Fram Strait (2015–2019; 5–100 m depth) to assess microbiome composition, substrate concentrations, and oceanographic parameters. Longitude and water depth were the major determinants (~30%) of microbial community variability. Bacterial alpha diversity was highest in lower-photic polar waters. Community composition shifted from west to east, with the prevalence of, for example, Dadabacteriales and Thiotrichales in Arctic- and Atlantic-influenced waters, respectively. Concentrations of dissolved organic carbon peaked in the western, compared to carbohydrates in the chlorophyll-maximum of eastern Fram Strait. Interannual differences due to the time of sampling, which varied between early (June 2016/2018) and late (September 2019) phytoplankton bloom stages, illustrated that phytoplankton composition and resulting availability of labile substrates influence bacterial dynamics. We identified 10 species clusters with stable environmental correlations, representing signature populations of distinct ecosystem states. In context with published metagenomic evidence, our microbial-biogeochemical inventory of a key Arctic region establishes a benchmark to assess ecosystem dynamics and the imprint of climate change.