Nanoparticles are the basic essential element of nanotechnology and it exhibits characteristic features based on their properties such as size, morphology and other size dependent properties. Their applications on agriculture, food and medicine are widely used nowadays. Agro-waste materials cause a global environmental problem worldwide. Maximizing the benefits of such waste material is a very important issue. The present investigation aimed to use nano-formulation techniques by using polyethylene glycol 8000 to produce nano-lycopene extracted from tomato waste (pomace) as well as evaluation the antioxidant, antimicrobial and anticancer activity of produced lycopene-NPs. The results of antioxidant activity using ABTS and DPPH assays revealed that nano-formulation of Lycopene increases the scavenging activity compared with lycopene. Also, both lycopene and lycopene-NPs showed antibacterial activity against all tested bacteria and antifungal activity against tested fungal species. MIC for lycopene and lycopene-NPs was determined. DNA damage induced by oxidative stress protection analysis indicated that lycopene-NPs has a great potential in DNA damage prevention compared to their respective controls. The MTT assay exhibited cytotoxic activity against three cancerous cell lines, MCF-7, HCT-116 and HepG2. As well as lycopene-NPs biosafety was tested against normal cell lines (BJ-1 and MCF-12F) compared to lycopene.