In this experimental study, the effect of pH (2, 4, 11), temperature (4, 85, 121 oC) and bile salt (0.3, 0.6, 1%) treatments on inhibitory potential of cell-free supernatant (CFS) obtained from barley sourdough dominant LAB (Pediococcus stilesii, Weissella cibaria, Lactobacillus brevis and Lactobacillus curieae) isolates, against B. subtilis were investigated based on microdilution method in comparison to untreated CFS. The safety of LAB isolates was also assessed by control of changes in serum parameters and liver enzymes in mice fed with them. Results were analyzed by one-way analysis of variance. Based on results, LAB CFS had antagonistic activity against B. subtilis. Inhibitory effects of the CFS under pH and temperature treatments were decreased, but under bile salt treatment was increased. Changes in serum parameters and liver enzymes in mice fed with LAB isolates were also in normal ranges and so on, safety of the LAB isolates was approved. By considering the antagonistic activity of LAB CFS under pH, temperature and salt treatments, against B. subtilis it is possible to use from the CFS as a biopreservative in food processing chain.