Aims: Folate (FA) is a B-vitamin that plays an important role in the prevention of several disorders. Although synthetic FA currently dominates the market, consumers tend to demand natural FA. Because microalgae can produce organic compounds photoautotrophically, we isolated and characterized FA-producing microalgal strains. Methods and Results: To isolate microalgae that produce vitamins de novo, fresh water samples were cultivated in a mineral salts medium without any vitamins. After repeated subculture, 11 isolates were obtained. A biological assay revealed that four isolates accumulated FA at significantly higher levels (15-36 mg kg-1 in dry biomass) than any known commercial microalgae. Thiamine content of the isolates was also remarkably high (71-90 mg kg-1 in dry biomass). Phylogenetic studies based on SSU-rDNA suggested that one isolate was Chlamydomonas reinhardtii, while others were likely novel species of Chlorococcum. Conclusion: There are no reports of toxicity associated with Chlamydomonas and Chlorococcum; therefore, the isolates are expected to be safe and useful not only as a promising alternative source of FA and thiamine but also as nutraceuticals for humans and animals. Significance and Impact of the Study: The present results advance our understanding of FA-producing microalgae in aquatic environments and suggest their potentials for application to biotechnological vitamin production.