Dissolved DNA (D-DNA) is operationally defined as DNA that passes through a 0.1 micrometer filter. It is composed of vesicles, viruses, and exocellular free DNA (F-DNA) and is ubiquitous in all aquatic systems, although the sources, sinks, and ecological consequences are largely unknown. Using a method that provides separation of vesicles, viruses, and F-DNA, we compared open ocean depth profiles of metagenomic DNA associated with each fraction. Pelagibacter-like DNA dominated the vesicle fractions for all samples examined over a depth range of 75–500 m. The viral DNA fraction was composed predominantly of myovirus-like and podovirus-like DNA, and contained the highest proportion of unannotated sequences. Euphotic zone F-DNA (75–125 m) contained mostly bacterial and viral sequences, with bacteria dominating in the mesopelagic (500–1000 m). A high proportion of mesopelagic (500 and 1000 m) F-DNA sequences appeared to originate from surface waters, including a large amount of DNA contributed by high-light Prochlorococcus species. Throughout the water-column, but especially in the mesopelagic, the composition of F-DNA sequences was not always reflective of co-occurring microbial communities that inhabit the same sampling depth. These results highlight the composition of F-DNA in different regions of the water-column (euphotic and mesopelagic zones), with implications for dissolved organic matter cycling and export (by way of sinking particles and/or migratory zooplankton) as a delivery mechanism.