Essential oils and Flavonoids are widely recognized for their beneficial effects in possessing many biological activities, such as antioxidant, antimicrobial and cytotoxic properties, also using in food additives and in the cosmetic industry. The aim of this work is to produce nanoparticles of hesperidin and essential oil extracted from waste orange peels, and study its biological impact compared to native products (hesperidin and essential oil) as antioxidant, antimicrobial and cytotoxic. Obtained products (nano-formulation) are characterized by zeta potential, electron scanning microscopy, spectrophotometric method and color analysis. The final products (nano-formulation) were examined as antioxidant agent by two assays (DPPH and ABTS analysis). The antioxidant activity of nano hesperidin and essential oil was evaluated and compared with none formulated. Nanoparticles of hesperidin and essential oil have a great potential in DNA damage prevention compared to their respective controls which were examined by DNA damage assay induced by oxidative stress protection analysis. The in vitro cytotoxic activity of nano formulated hesperidin and essential oil, towards (three cancerous cell lines, including: breast (MCF-7), colorectal (HCT-116) and liver (HepG2), and two non-cancerous control cell lines (fibroblast BJ-1and MCF-12F) compared with native product (essential oil and hesperidin) were examined using MTT assay. The hesperidin and essential oil NPs showed a positive effect on the inhibition of the proliferation of all tested cancer cell line. The antimicrobial activity of hesperidin, essential oil and their nano formulation form was evaluated using well diffusion assay. The nano formulation form of hesperidin and essential oil showed to improve the antimicrobial activity. These findings showed that nano-hesperidin and nano - essential oils play an important role as antioxidant, antimicrobial and cytotoxic agent's effects. These effects might be used for clinical trials and can represent driving formulation for novel chemotherapeutic agents and in food industry. Further studies about the therapeutic effects of nano-hesperidin and essential oil extracted from Egyptian orange peel are requested