Fusarium graminearum a causal agent of crown rot of wheat is an important soil-borne pathogen that causes yield reduction on cereal crops. Controlling this disease is crucial to ensure food security. The present study was conducted toward the research of biological method against this pathogen. Our research aimed to evaluate the antifungal activity of Citrus aurantium essential oil (EO) at different concentrations against F. graminearum in vitro. Also, we assessed the inhibitory effect of EO on disease development in greenhouse conditions. The results showed that EO exhibited a potent antifungal activity with no significant difference compared to chemical fungicide, Difenoconazole. The tested EO effectively inhibited radial growth of the pathogen in vitro, and it revealed significant decreasing on disease incidence from 91.7% to 32.8% in the in vivo bioassay. In addition, the seed treatment with C. aurantium EO improved significantly the growth parameters of the wheat seedlings compared to the untreated control. The chemical characterization of C. aurantium essential oil using the gas chromatography analysis allowed the identification of monoterpene hydrocarbon as major group of volatile compounds. These were Limonene (17.6%), Geranyl acetate (11.9%), Geranial (10.8%), Citral (10.3%), Linalool (9.9%) and Linalyl acetate (5.7%). From the current study, we can deduce that essential oils extracted from C. aurantium L. leaves can be used as an antifungal bio-product against phytopathogenic Fusarium species.