This study aimed to investigate the effects of temperature (4, 85, 121 °C), pH (2, 4, 11), and bile salt (0.3, 0.6, 1%)—as common conditions of gastrointestinal tract or thermal food processing—on antimicrobial activity of bacteriocin-like substances (BLS) obtained from barley sourdough lactic acid bacteria (LAB) against some of food-borne pathogens. In addition, the safety of LAB isolates was also evaluated. The isolates were identified by sequencing of PCR products as Lactobacillus brevis, Lactobacillus curieae, Pediococcus stilesii and Weissella cibaria. At low and high temperatures and pH values and at the presence of bile salt, BLS of the LAB isolates showed remarkable antimicrobial activity against studied indicator bacteria. Furthermore, BLS of L. curieae treated in pH 4 among all pH treatments, BLS of L. brevis and P. stilesii in 1% bile salt among all bile salt treatments, and finally BLS of L. curieae and P. stilesii treated at 4 °C among all temperature treatments were the most effective, respectively, against Aspergillus niger and A. flavus. The safety results demonstrated that consumption of LAB isolates had no adverse effects on general health of mice. Body weight of mice was increased by administration of LAB during 3 weeks, such as control group. Liver enzyme activities of mice treated with the LAB isolates had also insignificant difference with control mice.