The aim of the present study was to characterize the probiotic qualities and to study the efficacy of the selected isolates to produce folic acid by microbiological assay. Three isolates, IFM22B, IFM24B and IFM25B were selected for folate production in folic acid casei medium and the net folate yield was 59 ng/ ml, 56 ng/ml and 49 ng/ml, respectively. The three isolates showed more than 98 % similarity to Bacillus spp. by 16S rRNA sequencing. Spores of these strains showed excellent tolerance in partially simulated gastrointestinal tract conditions and exhibited antimicrobial activity against organisms such as Staphylococcus aureus, Klebsiella and Escherichia coli. Importantly, these isolates were susceptible to the most of the antibiotics tested, in conflict that they would not donate resistance determinants if administered in the form of probiotic preparations.