Secondary metabolites of bacteria associated with honey bees were evaluated as part of an investigation on their potentiality for apiary health. Low molecular weight compounds released into culture filtrates by the four bacterial isolates taken from surface of healthy honey bees were analyzed using time-of-flight mass spectrometry. Only one low molecular weight compound was found in the culture filtrate of each isolate.Bacillus thuringiensis, Bifidobacterium asteroidesandAcetobacteraceae bacterium, released into culture filtrates platynecine, a pyrrolizidine alkaloid of plant origin, which, until now, had never been reported as produced by bacteria.Lactobacillus kunkeeiproduced a 3,5-dinitropyridine, of unknown biological action never associated so far to bacteria. The highest relative concentration of platynecine was detected inB. thuringiensis(100%),B. asteroidesandA. bacteriumshowed a concentration above 50% and below 25% that concentration. An in vitro assay on the potential for controlling the parasitic miteVarroa destructorby the culture filtrates of the three platynecine-producing bacteria was performed. Varroa mite mortality was proportional to the platynecine relative concentration into culture filtrate. Although miticidal activity ofB. thuringiensismight be associated to other toxic proteins produced by this species,B. asteroidestoxicity towardV. destructoralong with the other findings of this study support the hypothesis that platynecine plays a direct or an indirect role in controlling varroa. Findings of this study suggest that secondary metabolites released by honey bee-associated bacteria represent a source of natural compounds to be considered in the challenge for counteracting the colony decline.