Twenty-seven honey samples from different floral sources and geographical locations were evaluated for their ability to inhibit the growth of seven food spoilage organisms (Alcaligenes faecalis, Aspergillus niger, Bacillus stearothermophilus, Geotrichum candidum, Lactobacillus acidophilus, Penicillium expansum, Pseudomonas fluorescens) and five foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Ser. Typhimurium, and Staphylococcus aureus) using an overlay inhibition assay. They were also tested for specific activity against S. aureus 9144 and B. stearothermophilus using the equivalent percent phenol test—a well diffusion assay corresponding to a dilute phenol standard curve. Honey inhibited bacterial growth due to high sugar concentration (reduced water activity), hydrogen peroxide generation, and proteinaceous compounds present in the honey. Some antibacterial activity was due to other unidentified components. The ability of honey to inhibit the growth of microorganisms varies widely, and could not be attributed to a specific floral source or demographic region produced in this study. Antibacterially active samples in this study included Montana buckwheat, tarweed, manuka, melaleuca, and saw palmetto. Furthermore, the bacteria were not uniformly affected by honey. Varying sensitivities to the antimicrobial properties were observed with four strains of S. aureus thus emphasizing the variability in the antibacterial effect of honey samples. Mold growth was not inhibited by any of the honeys tested. B. stearothermophilus, a heat-resistant spoilage bacteria, was shown to be highly sensitive to honey in both the overlay and well diffusion assays; other sensitive bacteria included A. faecalis and L. acidophilus. Non-peroxide antibacterial activity was observed in both assays; the highest instance was observed in the specific activity assay against B. stearothermophilus. Further research could indicate whether honey has potential as a preservative in minimally processed foods.