Abstract The Heterothalamus alienus oil was investigated in laboratory for control of different pests that affect the colonies of bees, Apis mellifera, against Varroa destructor mite, the bacterium that causes the American Foulbrood, Paenibacillus larvae and the fungus that produces the chalkbrood, Ascosphaera apis. The oil composition was analyzed by GC and GC/MS, the main components of the oil were Î²-pinene (44.4%) and trans-muurola-4(14),5-diene (9.2%). The concentration to kill 50% of the mites in 24 h (LC50) was 0.59 mg/cage. Inferior and superior limits were the following ones: 0.34 mg/cage and 1.01 mg/cage, LC50 was estimated for 48 h, and 72 h showed a slight increase with respect to the record of the 24 h. Paenibacillus larvae strains were Gram positive and catalase negative, the oil presented MIC values of 800â900 mg/L and MBC of 1000â1200 mg/L. Disks of impregnated flter paper with H. alienus oil around colonies of A. apis in growth inhibited the micelial growth signifcantly by 51% in the frst experiment (seven days) and by 31% in the second experiment (eight days).