It has been suggested that microorganisms present in honey are a potential source of antimicrobial compounds. This study aimed to isolate and characterize bacteria from 46 Greek honey samples of diverse botanical and geographical origin and to determine whether these bacteria demonstrate antibacterial activity against five important nosocomial and foodborne pathogens. In total, 2014 bacterial isolates were obtained and screened for antibacterial activity. Overall, 16% of the isolates inhibited the growth of Staphylococcus aureus, 11.2% inhibited the growth of Pseudomonas aeruginosa and Acinetobacter baumannii, 10.2% inhibited the growth of Salmonella Typhimurium and 12.4% of the isolates affected the growth of Citrobacter freundii. In total, 316 isolates that inhibited the growth of more than two of the tested pathogens were grouped by restriction fragment length polymorphisms (RFLP) analysis of the 16S rRNA gene amplicon. Fifty of them were identified by 16S rRNA gene sequencing. The majority, 62% of the isolates, belonged to the genus Bacillus. Only 10% of the isolates were identified as Gram-negative bacteria. Furthermore, in several bacterial isolates, genes encoding polyketide synthases and nonribosomal peptide synthetases that catalyze the biosynthesis of secondary metabolites which might contribute to the exerted antimicrobial activity, were detected. This study demonstrates that honey microbiota exerts antimicrobial activity and is a putative source of secondary metabolites against important nosocomial and food pathogens that warrants further investigation.