The volatile components from Croton campestris root bark were localized by an anatomical study and analyzed by gas chromatography–mass spectrometry for the first time. The roots of this plant showed secretory cells. These volatile constituents, isolated from the dichloromethane extract by hydrodistillation, were analyzed by gas chromatography–mass spectrometry. We found 69 components. They were characterized, and the major constituents of crude oil root barks were spathulenol (23.3%) and borneol (18.7%). Growth inhibitory activity of the active compounds in solution was evaluated by measuring minimal inhibitory concentrations using a broth micromethod. The minimal inhibitory concentration of root bark volatile constituents was 1.56 μg/mL for Staphylococcus aureus, 3.125 μg/mL for Candida albicans, and 6.25 μg/mL for Aspergillusniger.