The ECOMAR project was a multidisciplinary process study conducted in the mid-North Atlantic, coincident hydrodynamically with the Sub-Polar Front (SPF; 48–54°N) and topographically with Charlie-Gibbs Fracture Zone of the Mid-Atlantic Ridge, as part of the Census of Marine Life field project MAR-ECO. Midwater trawling was conducted during the 2007 and 2009 ECOMAR expeditions at 14 stations north and south of the SPF, day and night, in four discrete depth intervals from 0 to 1000 m. A total of 56 species of midwater fishes representing 44 genera and 18 families were collected, several of which are new records for the region and/or were not previously sampled during MAR-ECO sampling. An annotated species list with depth-of-capture data is provided. Three species of the genus Cyclothone (Cyclothone braueri, Cyclothone microdon and Cyclothone pallida) and the myctophid Benthosema glaciale combined to contribute ~88% of all specimens collected. This finding differs from results of previous net-based sampling in the same area, likely due to sampling scheme differences (diel sampling, upper 800 m concentration) and gear selectivity (mesh size, trawl speed). Quantitative data from ECOMAR midwater sampling and the previous 2004 G.O. Sars MAR-ECO expedition are compared. Despite differences in gear between the major MAR-ECO expeditions, abundance estimates of some dominant species were remarkably similar. Data showed that the SPF is an asymmetrical, taxon-specific biogeographic boundary for deep-pelagic fishes in the North Atlantic; the SPF is semi-permeable to some species in one direction, while a strong boundary for species in another direction. Deeper-living fish species did not appear as affected by the SPF as a boundary.