Plants have a wide range of active secondary metabolites that are frequently utilized to treat cancer. For the research, Dalbergia sissoo Roxb. ex DC. leaves (DS) were hydromethanolically extracted, and their phytochemical content was determined. Total phenolics and flavonoids were quantified along with the measurement of in vitro antioxidant and cytotoxic activities. The bioactive components in the extract were identified with the use of Gas Chromatography with High-Resolution Mass Spectrometry (GC-HRMS). The crude extract contained 177.500 ± 0.019 mg/ml of flavonoids and 296.122 ± 0.002 mg/ml of Gallic Acid Equivalent (GAE) phenolics. Moreover, plant crude extract showed significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity (IC50, 14.06 ± 0.18 μg/ml), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] activity (IC50, 25.97 ± 1.04 μg/ml), superoxide scavenging activity (IC50, 149.91 ± 0.39 μg/ml), and hydrogen peroxide scavenging activity (IC50, 133.37 ± 2.30 μg/ml). The thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) methods were used to confirm the presence of phenolic compounds. DS effectively scavenges nitric oxide. The crude extract of DS exhibited good cytotoxic effects on lung cancer cells (A549) with an IC50 value of 90.56 ± 2.32 (μg/ml), and less toxicity on normal lung cells (WI-38) with an IC50 value of 381.10 ± 1.58 (μg/ml). IC50 value of methotrexate (the standard drug) was 10.20 ± 1.82 μg/ml on A549 cells and 26.21 ± 1.14 μg/ml on WI-38 cells. Various staining techniques [4, 6-diamidino-2-phenylindole (DAPI), Acridine orange/Ethidium Bromide (AO/EB) staining, Giemsa staining] were used to determine the plausible mechanism of DS to induce apoptosis. The inhibiting mechanism of the crude extract was further demonstrated by the clonogenic assay and qualitative and quantitative measurements of Reactive Oxygen Species (ROS). Real-time polymerase chain reaction (RT-PCR) analysis revealed the induction of apoptosis in A549 cells through the activation of caspases 9 and 3 together with TRAIL receptors. In a nutshell, hydromethanolic extract of DS resulted in distinct apoptotic morphological alterations, ROS production, the initiation of apoptosis via activating the TRAIL receptors, caspase 9 and 3, and the suppression of colony formation in A549 cells.