Nitrogen (N2) fixation rates in the brackish Baltic Sea are among the highest per unit of area in the world. However, beyond the filamentous heterocyst-forming cyanobacteria, knowledge about the composition and distribution of N2-fixing microbes (diazotrophs) is limited. To address this, we investigated nitrogenase gene (nifH) composition and expression at coastal (<10 km offshore) and offshore (>10 km offshore) stations, at surface (avg. 1.8 m) and at depth (avg. 24 m) and in free-living (0.2–3.0 μm) and particle-associated size fractions (>3 μm). Surprisingly, nifH genes affiliated with Pseudanabaena and non-cyanobacterial diazotrophs (NCDs) dominated the composition whereas filamentous heterocyst-forming cyanobacteria accounted for almost 80% of the nifH transcripts. Salinity had a minor influence on the composition, but Aphanizomenon and Nodularia showed increased relative nifH gene expression at low and higher salinity, respectively. Pseudanabaena only accounted for up to 5% of the nifH transcripts and nifH gene expression by Candidatus Atelocyanobacterium thalassa (sublineage UCYN-A2) was mainly observed in the most saline western part of the Baltic. The only notable expression by NCDs (up to 15% of nifH transcripts at a given station) coincided with an upwelling event at the southern coast and was largely accounted for by a Pseudomonas-like nifH phylotype, recurrently found in the Baltic Sea. NCD relative abundances were dominant in coastal stations, presumably driven by sediment resuspension as evidenced by higher turbidity and DOC levels and the recovery of sediment diazotrophs in the pelagic zone. This study reveals the heterogeneity of the composition and activity of diazotrophs in the Baltic Sea, and underscores the need for future N2 fixation studies that include coastal and offshore Baltic waters.