Coastal microbial mats form a nearly closed micro-scale ecosystem harboring a complex microbial community. Previous DNA based analysis did not necessarily provide information about the active fraction of the microbial community because it includes dormant, inactive cells as well as a potential stable pool of extracellular DNA. Here we focused on the active microbial community by comparing 16S rRNA sequences obtained from the ribosomal RNA pool with gene sequences obtained from the DNA fraction. In addition, we aimed to establish an optimal and feasible sampling protocol that takes potential spatial and temporal heterogeneity into account. The coastal microbial mat investigated here was sampled randomly and at regular time points during one 24-h period. DNA and RNA was extracted and after conversion of the RNA fraction to cDNA, the V1-V3 and the V3-V4 regions of the 16S rRNA gene were targeted for high-throughput amplicon sequencing. We show that the community composition varies little in time and space whereas two amplified 16S regions gave significant different results. The largest differences were found when comparing the “resident community” (DNA) with the “active community” (cDNA/RNA); in the latter, Cyanobacteria dominated for almost 95% while they represented 60% of the resident fraction.