Propolis plays an important role in the exogenous defense of honeybee colony against pathogens. However, the studies dealing with the activity of propolis against bee pathogens are scarce. Poplar propolis extracts demonstrated promising activity against Paenibacillus larvae, the causative agent of American foulbrood. From the same propolis, five individual components and a mixture of caffeates were isolated, and their structures confirmed by spectroscopic data. Among the isolated propolis constituents are flavonoids, ferulic acid esters, and the oxylipin 9-oxo-10(E)-12(Z)-octadecadienoic acid, newly identified as propolis component. These substances were tested for their activity against P. larvae strains. The most active constituents were pinocembrin, 3-O-acetyl pinobanksin, and the caffeate mixture. This is the first communication of antimicrobial activity of individual propolis constituents against P. larvae; their important advantage is the fact that they are naturally present in the hive.