Marine fungi are important parasites and saprotrophs of organic material in the global oceans. However, only recently researchers revealed the important role of oceanic mycoplankton in the marine carbon and nitrogen cycle. Yet, basic parameters like the distribution of fungal biomass across major ocean basins, compared to other microbial groups considered to be important players in oceanic nutrient cycles, are lacking. Here, we collected oceanic fungal biomass from surface waters down to the abyssopelagic, covering both hemispheres in the Atlantic Ocean and crossing two oligotrophic gyres. By applying ergosterol extraction and fluorescence microscopy, as proxy for fungal biomass, to pelagic fungal cultures and environmental samples, we calculated novel biomass conversion factors that can be directly applied to future oceanic samples. Additionally, we analysed the fungal community structure by sequencing the ITS2 region. We found highest fungal biomass in productive surface regions, decreasing with distance to the coast, with distinct communities in productive and oligotrophic waters. Concisely, our findings indicate a considerable contribution of pelagic fungi to marine microbial biomass, which highlights again the necessity to integrate them in marine biogeochemical cycles.