<p>Tobacco black shank disease, caused by <i>Phytophthora nicotianae</i>, is one of the most devastating diseases affecting tobacco growth. Currently, chemical pesticides control is the most rapid and effective method for managing fungal diseases, yet it exerts significant impacts on the environment. Screening natural metabolites from plant that can prevent and control tobacco black shank disease is of great significance in sustainable development of tobacco cultivation. Quercetin is a natural flavonoid compound widely existing in plants and possesses various biological activities, such as antibacterial and antioxidant properties. However, its potential functions in controlling tobacco black shank has not been fully explored. In this study, we found that application of exogenous quercetin could significantly inhibit the growth of <i>P. nicotianae</i> and enhance tobacco resistance to <i>P. nicotianae</i> by enhancing tobacco antioxidant capacity, modulating the expression of cell wall biosynthesis, immune response and hormone signaling transduction related genes. In addition, we found that quercetin treatment could significantly induce the expression of <i>NtMYB4</i> to enhance plants resistance to <i>P. nicotianae</i>. Furthermore, we found that after inoculation with <i>P. nicotianae</i>, the accumulation of quercetin in the resistant variety was significantly higher than that in the susceptible variety, suggesting its important role in tobacco resistance to <i>P. nicotianae</i> in different varieties. Generally, our study provides new insights into the potential mechanisms by which quercetin promotes tobacco resistance to <i>P. nicotianae</i>, and and lays a theoretical foundation for future direct application of quercetin or breeding of high quercetin content varieties to prevent tobacco black shank disease.</p>