Bacterial strains isolated from US domestic honey were screened for antibacterial activity against Paenibacillus larvae ssp. larvae, the causative agent of American Foulbrood (AFB) in apiaries. A bacterial isolate (TH13) showing a high level of antimicrobial activity against P. larvae ssp. larvae ATCC 9545 was selected and identified as Paenibacillus polymyxa by 16S rRNA gene sequencing. The antimicrobial compound was purified by 80% saturated ammonium sulfate precipitation followed by CM-sepharose chromatography and reverse-phase HPLC. The molecular mass of the compound was determined to be 1168.78 Da by ESI-qTOF MS, matching that of polymyxin E1. The producer strain showed a broad range of antibacterial activity against gram-positive and -negative bacteria including P. larvae ssp. larvae ATCC 25747 and foodborne pathogens such as Bacillus cereus F4552 and Escherichia coli O157:H7 ATCC 43895. The selection of antibiotic-producing bacterial strains indigenous to honey as protective cultures against AFB may lessen the use of antibiotics in apiaries.