Fusarium head blight (FHB) is a devastating fungal disease of wheat caused by the fungus Fusarium graminearum that results in the substantial yield loss and grain contamination with mycotoxins. Currently, chemical fungicides remain the major component of FHB disease control, which carries risks to human and environmental health. Use of exogenous dsRNA to induce RNAi against pathogen’s genes, is an effective and sustainable approach for the control of FHB. In this study, we investigated the in-vitro and in-vivo effects of double-stranded RNA (dsRNA) application on growth and pathogenicity of Fusarium graminearum (Fg). We selected eight genes FgMGV1, FgRAS1, FgCOT1, FgPp2A, FgCAK1, FgTRI5, FgGMK1, and FgYCK1, that have previously been identified to have role in growth or pathogenicity of Fg. For the application, we produced and purified the dsRNA in laboratory using in-vivo methods along with graphene quantum dots (GQDs) as a nanocarrier. For the Fusarium assay studies, we collected Fg isolate from Indiana wheat fields in 2022. Growing Fg isolate in 1X synthetic low nutrient poor media without agar (SNA) media under different concentrations of dsRNA i.e., 1, 5, 10, 25 and 50 µg/mL showed a significant reduction in optical density evidenced at concentrations of 25 µg/mL and higher. These reductions in optical density were further validated by the regrowth experiments using fresh growth media. The performance of dsTRI5, dsMGV1, dsYCK1 and dsCOT1 in reducing fungal biomass in SNA medium was better than dsRNA of other genes, evidenced by lowering fungal biomass by almost half. The inhibitory effect of dsRNA on fungal growth in dsRNA incorporated Potato dextrose Agar (PDA) media was observed, showing distinct inhibition zones. When dsRNA was sprayed in the two wheat genotypes i.e., ‘AL105’ and ‘Gilat’ already inoculated with Fg, the percentage of symptomatic spikelets significantly reduced to 20-25% in both genotypes compared to 100% in controls. In addition, we also evaluated the effect of applied dsRNA on Fg infected spikes for lowering the accumulation of mycotoxin deoxynivalenol (DON) using gas chromatography–mass spectrometry (GC/MS) method. The results revealed lower accumulation and spread of DON in the neighboring spikelets of plants treated with dsRNA compared to no dsRNA treatment group. Furthermore, analysis of transcript abundance for two genes FgRAS1 and FgPP2A after the application of dsRNA in the liquid culture showed the gene expression reduction in treated Fg cultures by one-third as compared to untreated cultures.