Barleria, the third largest genus of the Acanthaceae family, carries a distinct position due to cultural as well as economic significance. Its aerial parts including stem, flowers, leaves, and underground part roots have been used in ancient civilizations as ornament, food, and for religious activities since ages. The presence of diverse phytochemicals is accountable for diverse healthcare applications including analgesic, antioxidant, and antimicrobial. In current work, previous knowledge is exploited as the foundation for the evaluation of Barleria longiflora leaves from the Siriya Kalvarayan hills region against lung cancer. The crude ethanolic extract of leaves has shown significant anti-inflammatory and antioxidant activity. Leaf extract has considerable in vitro free radical scavenging activity in DPPH and ABTS assay, i.e., 62% and 64%, respectively, in comparison to L-ascorbic acid (100 g.mL−1). Leaf extract has also shown commendable cytotoxicity against A549 cells (IC50: 71.00 μL.mL−1). Gas chromatography-mass spectrometry identified around 38 phytochemicals including stigmasterol, resorcinol, and 3,4-anhydro-d-galactosan. In silico analysis identified good binding molecules through molecular docking studies, especially stigmasterol having significant Ki values against the cancer receptors such as PI3K, mTOR, and ERβ. The phytochemicals from Barleria longiflora have shown commendable antimicrobial and anticancer activities which is also supported with in silico analysis. The compounds responsible for anticancer activity can become major ingredient for drug formulation after trials.