Benthic microalgae are key contributors to near-shore food webs and sediment stabilization. Temporal variability in microalgal biomass and production throughout the tidal cycle has been well documented; however, due to limitations of traditional methods of analysis patterns of community composition and diversity over such time scales have not been revealed. To explore the latter and better understand how short-term changes throughout the tidal cycle may affect community functioning, we compared benthic diatom composition and diversity over tidal stage shifts. We employed two disparate molecular techniques (denaturing gel gradient electrophoresis with Sanger DNA sequencing of excised bands and high-throughput community metagenome sequencing) to characterize diatom assemblages in representative muddy and sandy intertidal sites in Charleston Harbor, SC, USA. In support of prior studies, we found higher diatom diversity in sandbar as compared to mudflat sediments. Spatial differences were stronger relative to tidal temporal differences, although diversity metrics generally were highest after prolonged tidal immersion as compared to low-tide emersion or just after immersion at flood tide. Composition of the diatom assemblage differed markedly between sites, with species in genera Halamphora, Amphora, and Navicula dominating the sandbar, whereas Cyclotella, Skeletonema, and Thalassiosira were the most prevalent genera on the mudflat. Diatom composition differed by tidal stage, with assemblages during low-tide exposure distinct from samples taken after immersion. Both sandbar and mudflat sediments exhibited increases in relative proportion of epipelic diatoms and decreases in planktonic taxa during low-tide exposure. Our findings of short-term changes in species composition and dominance could inform primary productivity models to better estimate understudied diatom contributions in heterogeneous and highly variable tidal systems.