PLEASE NOTE, THESE DATA ARE ALSO REFERRED TO IN ANOTHER PUBLICATION. PLEASE SEE http://dx.doi.org/10.1098/rsos.160829. Dispersal limitation, not just environmental selection, plays an important role in microbial biogeography. The distance–decay relationship is thought to be weak in habitats where dispersal is high, such as in the pelagic environment, where ocean currents facilitate microbial dispersal. Most studies of microbial community composition to date have observed little geographical heterogeneity on a regional scale (100 km). We present a study of microbial communities across a dynamic frontal zone in the southwest Indian Ocean and investigate the spatial structure of the microbes with respect to the different water masses separated by these fronts. We collected 153 samples of free-living microorganisms from five seamounts located along a gradient from subtropical to subantarctic waters and across three depth layers: (i) the sub-surface chlorophyll maximum (approx. 40 m), (ii) the bottom of the euphotic zone (approx. 200 m), and (iii) the benthic boundary layer (300–2000 m). Diversity and abundance of microbial operational taxonomic units (OTUs) were assessed by amplification and sequencing of the 16S rRNA gene on an Illumina MiSeq platform. Multivariate analyses showed that microbial communities were structured more strongly by depth than by latitude, with similar phyla occurring within each depth stratum across seamounts. The deep layer was homogeneous across the entire survey area, corresponding to the spread of Antarctic intermediate water. However, within both the sub-surface layer and the intermediate depth stratum there was evidence for OTU turnover across fronts. The microbiome of these layers appears to be divided into three distinct biological regimes corresponding to the subantarctic surface water, the convergence zone and subtropical. We show that microbial biogeography across depth and latitudinal gradients is linked to the water masses the microbes persist in, resulting in regional patterns of microbial biogeography that correspond to the regional scale physical oceanography.