Bemisia tabaci is a serious pest in various horticultural crops in the world. The management of B. tabaci has been typically carried out by chemical pesticides. Due to the development of pesticide resistance and environmental contamination, however, it is necessary to develop alternative biopesticides using natural products from plants and natural enemies. Nicotiana benthamiana is a variety of wild tobacco plants and produce acyl sugars from glandular trichomes in the leaves. Acyl sugars are known to be highly toxic to various plant sapping insects such as whiteflies, aphids and thrips. Here, we extracted acyl sugars in two different ways from the leaves. At first, collected leaves were simply washed with water. Otherwise, collected leaves firstly dried and homogenized into the powder, then extracted with ethanol. Spray of 10% water-extracted solution into adult whiteflies showed 80% mortality. Otherwise, spray of 10% ethanol-extracted solutions showed complete mortality at 48 h after treatment and also strong repellency of adult whiteflies into the treated tomato plants. Our results suggest N. benthamiana is a useful for the control of whiteflies and can be used as an alternative natural pesticide for the whitefly management.