Natural antimicrobials have recently received more attention as means for addressing health concerns; however, most of these substances affect both probiotic bacteria and pathogenic bacteria. Therefore, natural antimicrobials which are specific to pathogenic bacteria and their applications within food systems require more investigation. This study evaluated antimicrobial effects of pomegranate peel aqueous extract (PPAE) on pathogenic bacteria including Escherichia coli, Salmonella enteric Typhimurium, Listeria monocytogenes, and Staphylococcus aureus, and two probiotics bacteria: Lactobacillus plantarum and Bifidobacterium lactis. Inhibition zone tests revealed excellent antimicrobial activity against the four tested pathogens while PPAE didn't inhibit probiotic bacteria. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were evaluated against the four pathogenic bacteria. Regarding to MIC values against tested pathogenic bacteria, different concentrations of PPAE ranging from 0.125 to 0.750 mg/mL were evaluated against the two probiotic bacteria. PPAE showed a protective effect on the two tested probiotics. HPLC analysis of PPAE revealed that catechin, protocatechuic, and rutin were the most abundant phenolic compounds. Adding PPAE at MIC concentration to soft cheese reduced E. coli by almost 2 log compared with controls, while B. lactis count was boosted after 30 days of cold storage. Moreover, the total viable and psychrotrophic bacteria counts were decreased compared with controls, and the combination of probiotic bacteria and PPAE improved the overall acceptability of the cheese. This study confirmed that using PPEA can suppress the growth of E. coli in cheese supplemented with probiotic bacteria and enhances shelf life. Furthermore, PPAE increased the antioxidant capacity of cheese.