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Lactic acid bacteria and their own bacteriocin are great promising in health and industry; hence, too many lactic acid bacteria species and metabolites are involve in health maintenance and treatment of infectious disease as well as food preservation and dairy production. Current study sought to produce, isolate, purify and characterize novel bacteriocin from oral Bifidobacterium adolescentis and studying its effect on antibiotic-resistant pathogenic bacteria. Saliva of 18 volunteers postmenopausal women with their age ranging from 49 to 58 years was included. Bifidobacterium spp. cultivates in anaerobic conditions and identified by API50. Bifidobacterium bacteriocin prepared in De Man, Rogosa and Sharpe broth (MRS) broth as a crude preparation then concentrated by ammonium sulphate precipitation and purified by ion exchange and gel filtration chromatography. The molecular weight of B. adolescentis bacteriocin was estimated by gel filtration chromatography. The effect of temperature and pH value on purified bacteriocin activity was estimated using Escherichia coli American Type Culture Collection (ATCC) and Staphylococcus aureus ATCC. To determine the nature of produced bacteriocin and whether or not the bacteriocin contain lipid or carbohydrate moiety, it was treated with some enzymes (pepsin, lipase, α-amylase, papain and chemotrypsin). Challenged pathogenic bacteria were surveyed for occurrence of antibiotic resistance genes by PCR and to verify the inhibitory effect of B. adolescentis bacteriocin on challenged pathogenic bacteria, disc diffusion method was used. The result of anaerobic culture showed that, 35 Bifidobacterium isolates were obtained, the predominant species was B. adolescentis other species was lesser, namely Bifidobacterium dentium, Bifidobacterium longum and Bifidobacterium urinalis. The purified B. adolescentis bacteriocin was 14 600-Da protein and was active at wide range of pH value (3–8), thermostable and has no lipid or carbohydrate moiety. Almost all of pathogenic bacteria, whether or not they carry antibiotic resistance genes, appeared to be sensitive to crude and purified bacteriocin but purified bacteriocin was more effective as antimicrobial agent. The results suggest that Bifidobacterium spp. was dominant in oral cavities of postmenopausal women who have no caries it may prevent dental caries via antimicrobial activities of their own bacteriocin. Bifidobacterium bacteriocin is protein in nature, without lipid or carbohydrate moiety, heat-stable and active at wide range of pH values and can be classified as type II bacteriocin and so-called bifidoadocin. The antimicrobial activity of bifidoadocin makes it probable food preservative and antagonistic agent against pathogens of oral cavity and causative agents of dental caries as well as other pathogens.