The aim of this study was to explore the phytochemical profile, in vitro free radical scavenging and in vitro cytotoxicity effects of ethyl acetate extract of Nerium odorum. The qualitative analysis of the phytocompounds of Nerium odorum was performed by Spectrophotometry method. Its antioxidant activity was analyzed by the method of DPPH-radical scavenging activity and bleaching of β-carotene. The in vitro cytotoxicity of ethyl acetate extract of Nerium odorum evaluated by WST-1 assay on normal and HT-29 human colorectal adenocarcinoma cell line and MDA-MB231 metastatic mammary adenocarcinoma cell lines. Medicinal plants have been used for many human diseases such as cancer, heart diseases, arthritis, etc. Thus, in this study we have investigated the phytochemical profile of ethyl acetate extract of Nerium odorum such as preliminary screening, HPTLC quantification, GC-MS and FT-IR analysis. The ethyl acetate extract of Nerium odorum clearly possesses significant antioxidant potential due to its rich phytochemicals.