B9 vitamin (folic acid) and its derivatives (folates) are essential vitamins for a healthy body and could be found in various food matrixes but also in drinks like beer. This work aims to determine the impact of fermentation conditions of brewing yeast on folate production. Influences of various parameters were considered: type of yeast (Saccharomyces cerevisiae and Saccharomyces pastorianus) in optimal laboratory conditions (stirred wort at 30 °C), industrial working conditions (static wort at 12 or 18 °C), addition of fermentation activators (yeast peptide complex and oleic acid) and ethanolic stress (4%, v/v). Total content, production and productivity of folates were noticeably increased with activators. On the contrary, ethanolic stress slowed down yeast metabolism so as folates production. Independently apart from the applied fermentation condition, folate synthesis were quite similar for the different strains tested, allowing us to the conclusion that yeast type seems to have negligible influence on vitamin profiles.