In the aim of selecting starter cultures, thirteen species of Bacillus spp. including six Bacillus subtilis ssp. subtilis, four Bacillus licheniformis and three Bacillus amyloliquefaciens ssp. plantarum isolated from traditional Bikalga were investigated. The study included, for all isolates, genes, determination of minimal inhibitory concentration (MIC) for 24 antimicrobials and detection of resistance by PCR using specific primers. The isolates were also examined for their resistance to pH 2.5 and their tolerance to 0.3% bile over 4 h. Results showed that most studied isolates, in particular B. subtilis ssp. subtilis G2, H4, C6, I7 and B. amyloliquefaciens ssp. plantarum A4, I8, G3 were susceptible to most antimicrobials tested while all B. licheniformis isolates showed high resistance level. The resistance observed towards the antimicrobials (chloramphenicol, erythromycin, kanamycin, penicillin, streptomycin and trimethoprim) in this study may be intrinsic as no positive amplicon was observed for the most prevalent resistance genes investigated (catpIP501, erm(A), erm(B), erm(C), aph(3”)-I, aph(3”)-III, ant(2”)-I, blaZ, aadA, aadE, StrA, StrB, dfr(A)). Furthermore, based on their good survival in pH 2.5 and in 0.3% bile all the tested isolates may be able to resist passage through the gastro-intestinal tract conditions. Regarding these results, isolates G2, C6, I7, H4, A4, I8 and G3 may be useful as starter cultures to optimize Hibiscus sabdariffa seeds fermentation into Bikalga.