Background <p>Human fibrosarcoma is an aggressive soft tissue malignancy with limited targeted therapies, that prone to rapid metastasis and chemotherapy resistance. Targeting prooxidant pathways represents a promising clinical strategy for the control of fibrosarcoma.</p> Objective <p>This study evaluated the anti-cancer efficacy and underlying molecular mechanism of 4-chloro-7-Nitrobenzofurazan (NBD) in human fibrosarcoma HT-1080 cells.</p> Methods <p>Cell viability was determined by MTT assay in HT-1080 cells and normal lung fibroblast (IMR-90). Apoptotic populations and cell cycle progression were determined by flow cytometry using Annexin V-FITC/PI and propidium iodide (PI). Intracellular ROS levels were analyzed using DCFDA. Signaling pathways were assessed through p53/p21 luciferase reporter assay and Western blotting and validated using antioxidant control (Glutathione; GSH) and genetic control (p53 knockout HT-1080).</p> Results <p>NBD selectively decreased HT-1080 cell viability (IC₅₀ – 1.83 µM) while sparing the IMR-90 cells (IC₅₀ – 34.02 µM). Flow cytometry analysis revealed concentration-dependent induction of late apoptosis and sub-G₀/G₁ phase DNA fragmentation. Mechanistically, NBD generated intracellular ROS and suppressed the catalase expression, disrupting the redox homeostasis. This oxidative stress induced a biphasic p21 response with potent p53 activation that shifted the cells from cytostatic arrest to apoptosis. Consequently, BCL-2 family proteins shifted toward the pro-apoptotic path, triggering executioner cleaved caspase-3 activation. ROS scavenging by GSH completely blocked p53 upregulation and caspase-3 cleavage, restoring cell viability. Furthermore, p53 KO completely diminished the NBD mediated caspase-3 activation and restored the cell survival.</p> Conclusion <p>NBD triggers selective apoptosis in HT-1080 cells via the ROS-induced p53 pathway, highlighting its potential as a redox-targeting therapeutic candidate.</p>