Declining coral reef health has triggered a multitude of studies focusing on the diversity and functional roles of reef-associated microbial communities. However, comparatively little is known about the microbial communities in deeper sediment assemblages (> 1 m) below the reef substrate. Here, we present the first characterisation of microbial community structure in sub-bottom sediment of up to 3.5 m beneath the reef substrate. Cores were retrieved from three reef sites in Singapore and profiled via 16S rRNA (V4 region) gene sequencing. Despite each core containing a large number of unique ASVs, cores from the same reef showed more similar taxonomic profiles compared to cores from other reefs. We detected limited differences in diversity between either cores or sediment facies. Our results provide novel insight into microbial communities in sub-bottom reef sediment and contribute to the growing research on reef–microbial interactions.