Lactuca Sativa L., known for its ethnomedicinal applications, boasts a plethora of medicinal claims, yet remains largely underexplored. Traditionally, various parts of the plant have been utilized for treating ailments such as skin disorders and leprosy. This study aims to comprehensively investigate the qualitative, quantitative, and antifungal aspects of Lactuca sativa L. Different tests were conducted to ascertain both qualitative and quantitative parameters, including the presence of protein, glycosides, alkaloids, carbohydrates, and terpenoids. The total ash and acid insoluble ash values were determined to be 12.8% and 10.85%, respectively, providing insight into the mineral composition of the plant material. Furthermore, extractive values for various solvents, such as alcohol and water, were measured at 12.43% and 17.73%, respectively, indicating the solubility of different constituents. Moisture content, calculated through the loss on drying method, yielded a value of 1.5%, crucial for understanding stability and storage requirements. The pH value, determined with a digital meter, was recorded at 6.994, revealing the acidic or alkaline nature of the plant extract. Preliminary phytochemical screening unveiled that aqueous and methanolic extracts serve as rich sources of amino acids, flavonoids, and proteins. Additionally, the water extract exhibited the presence of carbohydrates and saponins, expanding the repertoire of bioactive compounds present in Lactuca sativa L. High-Performance Thin-Layer Chromatography (HPTLC) analysis of the hydroalcoholic extract revealed the presence of three peaks at different Rf values at 254 nm, indicating the presence of specific compounds with distinct polarities and absorbance characteristics. Similarly, five peaks were observed at 366 nm, highlighting the complex chemical profile of the extract. In conclusion, this study provides valuable insights into the qualitative, quantitative, and antifungal aspects of Lactuca sativa L., laying the groundwork for further exploration of its medicinal properties and potential pharmacological applications. Further research is warranted to isolate and identify the bioactive compounds responsible for observed pharmacological activities and validate the traditional uses of this ethnomedicinal plant.