Background A remarkable range of environmental conditions is present in the Hawaiian Islands due to their gradients of elevation, rainfall and island age. Despite being well known as a location for the study of evolutionary processes and island biogeography, little is known about the composition of the non-marine algal flora of the archipelago, its degree of endemism, or affinities with other floras. We conducted a biodiversity survey of the non-marine macroalgae of the six largest main Hawaiian Islands using molecular and microscopic assessment techniques. The present biodiversity survey aimed to evaluate the competing ideas of whether endemism or cosmopolitanism better explain freshwater algal distribution patterns, and to provide a baseline data set for monitoring future biodiversity changes in the Hawaiian Islands. Results A total of 1,786 aquatic and terrestrial habitats and 1,407 distinct collections of non-marine macroalgae were collected from the islands of Kauai, Oahu, Molokai, Maui, Lanai and Hawaii from the years 2009-2014. Targeted habitats included streams, wet walls, high elevation bogs, taro fields, ditches and flumes, lakes/reservoirs, cave walls and terrestrial areas. Numerous collection sites lacked freshwater macroalgae, and these sites were typically terrestrial and wet wall habitats that were sampled for diatoms and other microalgae. Approximately 50% of the identifications were of green algae, with lesser proportions of diatoms, red algae, cyanobacteria, xanthophytes and euglenoids. Molecular characterization of individual isolates yielded 898 DNA sequences representing eight different markers, which enabled an assessment of the number of taxonomic entities for genera collected as part of the survey. Forty-four well-characterized taxa were compared with literature records for these same taxa to assess global distribution patterns, and were characterized as belonging to one of four distributional categories based on how many regions of the world in which they were reported as being present. This analysis revealed no clear biogeographic affinities of the flora, with 27.3% characterized as “cosmopolitan”, 11.4% “endemic”, and 61.3% as intermediate. Conclusions The Hawaiian freshwater algal biodiversity survey (2009-2014) represents the first comprehensive effort to collect, characterize and document the non-marine algae of a tropical region in the world using both morphological and molecular tools. The survey supported the design and population of the Hawaiian Freshwater Algal Database, which houses all project data and serves as a digital repository of photographs and micrographs, georeferenced localities and DNA sequence data. These analyses yielded an updated checklist of the non-marine macroalgae of the Hawaiian Islands, and revealed varied biogeographic affinities of the flora that are likely a product of both natural and anthropogenic dispersal.