Abstract Biological activity was evaluated of the Tagetes minuta essential oil in different in vitro laboratory experiments on the mite (Varroa destructor), honeybees (Apis mellifera), the bacterium that causes the American Foulbrood (Paenibacillus larvae), and the fungus that produces chalkbrood (Ascosphaera apis). Two methods of complete exposure were used for mite lethality test: by spraying in Burgerjon's tower with 10 mg of active ingredient in solution with distilled water and emulsion, and in unmodified Petri dishes (60 x 20 mm) with oil (different concentrations) diluted in 1 mL of ethanol. Ratio selection was obtained as: LD50 of Apis mellifera/LD50 of V. destructor. Determination of Minimal Inhibitory Concentrations (MIC): to P. larvae was tested at concentrations of 25, 50, 100, 150, 200, 250, 350, 450, 500, 600, 650, 700, 800 and 1000 ppm. An A. apis strain was grown on agar MY20 supplemented with variable concentrations (between 0â800 ppm) of T. minuta oil was evaluated. Results obtained in tests of total exposure showed that the concentration was able to kill 50% of mites in 24 h (DL50) and was estimated to be 4.37 mg/cage. The efficacy after spray treatment reached 56%. The ratio selection was 3:11. Against P. larvae, the oil showed MIC values ranging from 700â800 Î¼L/L depending on the tested bacterial strains. Tagetes minuta oil in agar MY20 inhibited mycelial growth of A. apis above concentrations of 200 ppm (p = 0.0001). Oil concentrations of 700 and 800 ppm achieved maximum growth inhibition of A. apis (67% of growth inhibition on average). Tagetes minuta oil demonstrated in vitro antibacterial, antifungical and miticide activity, although this oil shows a moderate inhibitor effect compared with other essential oils of native plants from Argentina. However, this oil presents a ratio selection that would allow it to be used in field conditions with a good safety margin. It is possible that this oil can be used in combination with others, in integrated pest management strategies in bee colonies. Key Word Index: Tagetes minuta Asteraceaebioactivity Apis mellifera honeybee pathogens Ascosphaera apis Paenibacillus larvae parasitic bee mite Varroa destructor essential oil composition(Z)-Î²-ocimene(Z)-ocimenone