Honey bee hygienic behavior is an economically important mechanism of resistance against brood disease. Colonies selected for hygienic behavior detect, uncap and remove diseased brood from the colony before it reaches an infectious stage, thus reducing disease transmission. Hygienic bees have greater olfactory sensitivity to the odor of diseased brood compared to non-hygienic bees, but the volatile compounds that elicit the behavior have not yet been determined. The goal of this study was to determine which compounds associated with diseased brood the bees detect and to assay whether the application of these compounds to healthy brood would elicit the motor tasks of uncapping and removal of the brood. Volatile compounds were collected from healthy honey bee larvae and those infected with chalkbrood Ascosphaera apis. The volatiles were analyzed by mass spectrometry and the chemical profiles compared to determine which compounds were unique to chalkbrood infected larvae. Gas chromatography-electroantennographic detection (GC-EAD) determined that three compounds unique to diseased larvae were detected by adult honey bees: benzyl alcohol, phenyl ethanol and phenethyl acetate. A field bioassay was used to determine differences between bees bred for hygienic and non-hygienic behavior in their ability to detect and remove healthy larvae treated with disease compounds. In the future, beekeepers may use this bioassay to select their most hygienic colonies for breeding.