Traditional dairy products are the main source of probiotic microorganisms. Hence, this study aimed to evaluate the safety and antimicrobial potential of Lactobacillus pentosus 22C, isolated from traditional yogurt from Kermanshah province, Iran. Strain 22C showed no undesirable amino acid decarboxylase and β-hemolytic activities. Virulence associated genes were detected in the isolate. The strain produced an antimicrobial molecule named pentocin 22C, a small peptide with a relative mass between 5 and 10 kDa. Bacterial inhibition was pH-independent, with greater activity at pH 4–6. Purified or semi-purified antimicrobial agents can be used as bio-preservatives, and the producing strain can be used as a starter culture to improve food safety. Therefore, a biosafety assessment should be conducted. L. pentosus 22C, isolated from a traditional dairy product, has antimicrobial ability, although its safety must be evaluated.