Bemisia tabaci is a serious pest in various horticultural crops in the world. Due to use of chemical pesticide for their management they develop pesticide resistance and environmental contamination. It is necessary to develop alternative bio-pesticides 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. When adult whiteflies were reared with fresh N. benthamiana leaves, they were completely dead within 84 h. Oral feeding of 20% N. benthamiana extracts using ethanol and water showed complete mortality of whiteflies within 48 hours. Spray of N. benthamiana extracts into the leaves was lethal to eggs but not to nymphs of whiteflies. Further, tomato plants sprayed with N. benthamiana extracts were highly repellent to adult whiteflies. Quantitative real-time PCR indicated that the expression of various genes of B. tabaci was changed by oral feeding of N. benthamiana extract. This study suggests N. benthamiana extract is a useful for the control of whiteflies and can be used as an alternative natural pesticide for the whitefly management.