SummaryThis study selected the best folate‐producing strains in folate‐free medium among five commercial starter and probiotic strains and evaluated the utilisation of fruit by‐products (pitaya, passion fruit and grape), modified pectin from orange albedo (MP) and other carbohydrate sources for growth and folate production by the selected strains. The three selected strains [Lacticaseibacillus (L.) rhamnosus GG, Bifidobacterium (B.) infantis BB‐02, and Streptococcus (S.) thermophilus TH‐4] grew equally well in m‐MRS broth containing these products or carbohydrates, reaching populations above 7 log CFU mL−1 in 24 h. Production of folate was strain‐dependent. L. rhamnosus GG showed the highest folate production when supplemented with fruit by‐products and MP, achieving the utmost folate concentration after 24 h in the presence of grape by‐product (101.2 ± 11.2 ng mL−1). The MP has also influenced positively the production of folate by this strain. The study evidenced that fruit by‐products can be explored as substrates for bio‐enriched food product development using selected folate‐producing strains.