Complete eradication of aggressive head and neck squamous cell carcinoma (HNSCC) still remains a major challenging problem due to numerous resistance properties of cancer stem cells (CSC) which is crucially responsible for tumor recurrence and metastasis. This challenge causes a high demand for the emergence of novel targeted treatment modalities for improved therapeutic efficacies. Phytochemicals derived from plants proves to be a wide reservoir of important drug candidates which have the potential to impede multiple aspects of malignant growth and progression. In the present study, we aimed to synthesize gold nanoparticles in a rapid and cost-effective manner by utilizing Madhuca indica flower extract and to evaluate its anticancer efficacy on head and neck cancer model via targeting cancer stemness and EMT. The phytochemicals present in the Madhuca indica flower extract acted as an effective reducing agent helping in the green synthesis of gold nanoparticles. The generated AuNPs were characterized by UV-Vis spectroscopy, XRD, FTIR, TEM, FE-SEM, DLS, EDX. Anti cancer potential of synthesized AuNPs were evaluated by in vitro and ex vivo HNSCC model. In vivo toxicity was assessed in Swiss albino mice model. The gold nanoparticles were characterized using UV-Vis spectroscopy which revealed unique wavelength maxima at 550 nm and its crystalline nature was confirmed by XRD. AuNPs were observed to be spherical in shape with the mean diameter of 20.34 ± 4.36 nm and zeta potential of nearly -50 mV. The FTIR spectral shift indicated the incorporation of various functional groups. MI-AuNP depicted strong anticancer attributes against HNSCC cell lines SCC154 and FaDu through significant inhibition of cancer stemness and EMT as evident from decreased tumor sphere forming efficiency and CD44+/CD24- subpopulation along with dose dependent downregulated expression of relevant CSC markers and EMT markers both in vitro and ex vivo HNSCC model. Additionally, no evidence of in vivo toxicity has been observed with MI-AuNP administration. In conclusion, this study reported for the first time that the MI-AuNP synthesized by novel green chemistry can efficiently prevent the self-renewal capability of HNSCC by targeting Cancer stemness. The scientific significance of this study lies in the fact that MI-AuNP might be a novel and potential therapeutic candidate against aggressive and metastatic HNSCC. The findings in this study unravels the way for developing a novel therapeutic candidate against aggressive and metastatic HNSCC with a much higher prognostic potential and significantly reduced off target toxicity.