Background: The increase of multidrug-resistant pathogens causes increased concerns about the efficacy of conventional treatment, which requires the search for new alternatives to antibiotics. The present study aimed to evaluate the antibacterial activity of lactobacilli isolated from eight (n=8) typical Apis mellifera honey produced in Mostaganem region against four pathogenic bacteria responsible of bovine mastitis. Methods: Lacbacilli strains, isolated from different media were firstly presumptively identified by catalase test, Gram staining and cells morphology, than confirmed by partial 16S rRNA sequence analysis. Antibacterial activity of isolates was determined by the overlay agar method. Antibiotic susceptibility of mastitis pathogens was tested by disk diffusion technique. Result: Two species of lactobacilli were isolated and identified as Lactobacillus plantarum and Lactobacillus pentosus. Antibiotic sensitivity test demonstrated that mastitis pathogens showed the resistance to antibiotics tested. All the isolates showed antimicrobial activity with inhibition zone diameters ranged from 5±0.02 to 21±2.12 mm. The results revealed that honey can be considered as a source of Lactobacillus with antibacterial activities which can be used as alternative treatment of bovine mastitis.