In intertidal sediments, circadian oscillations (i.e., tidal and diel rhythms) and /or depth may affect prokaryotic activity. However, it is difficult to distinguish the effect of each single force on active community changes in these natural and complex intertidal ecosystems. Therefore, we developed a tidal mesocosm to control the tidal rhythm and test whether diel fluctuation or sediment depth influence active prokaryotes in the top 10 cm of sediment. Day- and night- time emersions were compared as they are expected to display contrasting conditions through microphytobenthic activity in five different sediment layers. A multiple factor analysis revealed that bacterial and archaeal 16S rRNA transcript diversity assessed by pyrosequencing was similar between day and night emersions. Potentially active benthic Bacteria were highly diverse and influenced by chlorophyll a and phosphate concentrations. While in oxic and suboxic sediments, Thaumarchaeota Marine Group I (MGI) was the most active archaeal phylum, suggesting the importance of the nitrogen cycle in muddy sediments, in anoxic sediments, the mysterious archaeal C3 group dominated the community. This work highlighted that active prokaryotes organise themselves vertically within sediments independently of diel fluctuations suggesting adaptation to physicochemical specific conditions associated with sediment depth.