The diazotrophic community in microbial mats growing along the shore of the North Sea barrier island Schiermonnikoog (The Netherlands) was studied using microscopy, lipid biomarkers, stable carbon (δ13CTOC) and nitrogen (δ15N) isotopes as well as by constructing and analyzing 16S rRNA gene libraries. Depending on their position on the littoral gradient, two types of mats were identified, which showed distinct differences regarding the structure, development and composition of the microbial community. Intertidal microbial mats showed a low species diversity with filamentous non-heterocystous Cyanobacteria providing the main mat structure. In contrast, supratidal microbial mats showed a distinct vertical zonation and a high degree of species diversity. Morphotypes of non-heterocystous Cyanobacteria were recognized as the main structural component in these mats. In addition, unicellular Cyanobacteria were frequently observed, whereas filamentous heterocystous Cyanobacteria occurred only in low numbers. Besides the apparent visual dominance of cyanobacterial morphotpyes, 16S rRNA gene libraries indicated that both microbial mat types also included members of the Proteobacteria and the Cytophaga–Flavobacterium–Bacteroides group as well as diatoms. Bulk δ15N isotopes of the microbial mats ranged from +6.1‰ in the lower intertidal to −1.2‰ in the supratidal zone, indicating a shift from predominantly nitrate utilization to nitrogen fixation along the littoral gradient. This conclusion was supported by the presence of heterocyst glycolipids, representing lipid biomarkers for nitrogen-fixing heterocystous Cyanobacteria, in supratidal but not in intertidal microbial mats. The availability of combined nitrogen species might thus be a key factor in controlling and regulating the distribution of the diazotrophic microbial community of Schiermonnikoog.