Aims: Nineteen strains of bifidobacteria were screened for main folate forms composition in synthetic folate free and complex folate-containing media. Methods and Results: HPLC was used to analyze deconjugated folates extracted from bacterial biomass. Most strains had a total folate content above 4000 μg/100g DM. The highest value of 9295 μg/100g DM was found in B. catenulatum ATCC 27539 and the lowest in B. animalis subsp. animalis ATCC 25527 containing 220 μg/100g DM. 10 strains grew in synthetic folate free medium (FFM), showing folate autotrophy and suggesting folate auxotrophy of the remaining nine. In the autotrophic strains, a consistently higher folate level was found in FFM as compared to a more complex folate-containing medium, suggesting reduced requirements for folates in the presence of growth factors otherwise requiring folates for synthesis. The contents of total folate, 5-CH3-H4folate and H4folate were strain dependent. 5-CH3-H4folate dominated in most strains. Conclusions: Our results show that bifidobacteria folate content and composition is dynamic, strain-specific and depends on medium. Suitable selection of the growth conditions can result in high levels of folate per cell unit biomass. Significance and Impact of Study: This suggests that certain bifidobacteria may contribute to the folate intake; either directly in foods, such as fermented dairy products, or in the intestine as folate-trophic probiotics or part of the natural microbiota.