Growth of Gram-positive bacteria was more strongly inhibited compared with that of Gram-negative bacteria when cultured in media containing 1,5-anhydro-D-fructose (AF). For example, the growth of Escherichia coli ML-35 was barely inhibited, with all of the AF in the medium being metabolized after being converted into 1,5-anhydro-D-glucitol (AG), whereas the growth of Staphylococcus aureus ATCC 10832 was strongly inhibited, with most of the AF being retained and only low amounts of AG being observed. Because we detected NADPH-dependent AF reductase (AFR; AG-forming) in the cell extract of both these strains, we assumed that the difference in the ability to regenerate NADPH was the main factor responsible for growth inhibition in the presence of AF. The N-terminal amino acid sequence of AFR isolated from E. coli ML-35 was consistent with that of 2,5-diketo-D-gluconate reductase, with sequences characteristic of the aldo-keto oxidoreductase family. We also found that growth of Listeria monocytogenes ATCC BAA-679 was inhibited by addition of AF and the growth of this organism was further inhibited by a combination of AF and sodium chloride. Furthermore, AF inhibited spore formation and germination in Bacillus subtilis NBRC 3009. These results demonstrate that AF is a beneficial material for the food industry.