We investigated dinitrogen (N2) fixation activity and diazotroph community composition across the Cape Verde Frontal Zone (CVFZ), from photic epipelagic waters (0-200 m) to aphotic meso- (200–1000 m) and bathypelagic (> 1000 m) waters. The highest N2 fixation rates of 4.1 ± 2.2 nmol N l− 1 day− 1 and 7.8 ± 2.3 nmol N l− 1 day− 1 were in epipelagic waters south of the front. We detected aphotic N2 fixation in 5 out of 32 samples, primarily south of the front, and sporadically down to 3,000 m, with rates ranging from 0.03 ± 0.01 nmol N l− 1 day− 1 to 0.07 ± 0.01 nmol N l− 1 day− 1. Cyanobacteria dominated the diazotroph community and nitrogenase gene (nifH) expression profiles in surface waters and, surprisingly, in aphotic waters. The detection of cyanobacterial nifH genes by DNA sequencing and quantitative PCR in the aphotic zone, together with nifH expression in meso- and bathypelagic waters, indicates a downward flux of metabolically active cyanobacteria, and points to a contribution to the observed aphotic N2 fixation rates. In the photic zone, UCYN-A dominated north of the front, whereas Trichodesmium was mainly found in the southern region. However, our results also show that cross-frontal advection of cyanobacterial diazotrophs can occur via intrusions of surface water. Salinity, temperature, and mixed layer depth were the main determinants of the diazotroph composition and distribution of the key cyanobacteria. Thus, the front appeared to act as a dynamic barrier controlling the distribution of cyanobacterial diazotrophs.