Abstract: In this study, the GC/MS analysis and in vitro screening of antioxidant antimicrobial and antitumor activities of essential oil extracted from Oleander flower (Nerium oleander) were investigated. The GS/MS analysis recorded 64 components account 94.69% of the total essential oil were composition. Amount of total phenolics was 136.54±3.32 mg as gallic acid/g essential oil. The antioxidant activity was studied by three methods (DPPH assay; β-Carotene/linoleic acid a bleaching assay and ferric reducing power assay). Oleander essential oil had significantly antioxidant activity compared to synthetic antioxidants (trolox and BHT). Antitumor activity was tested as ability of inhibition the growth of ehrlich ascites carcinoma cells line and obtained result indicated gradually increase of antitumor activity with increasing of oil concentration. Antimicrobial activity was observed by agar disc diffusion technique against different strains of Gram-positive; Gram-negative; yeast and mold. The essential oil displayed a variable degree of antimicrobial activity against the different strains tested compared with that of the standard antibiotics tested and the Minimum Inhibition Concentration (MIC) values ranged from 125 to 500 and 250 to 2000 μg mL-1 for bacteria and fungi respectively. The toxicity of oleander essential oil was studied in animal model system by different parameters including LD50 ; diarrhea; (GPT) activity; lactate dehydrogenase (LDH) activity and createnine levels. Toxicity results indicated that no adverse effect recorded with all concentrations of oleander oil range used in present study.