Onosma bracteata Wall. (Boraginaceae family) is one of the important constituents of Ayurvedic drugs which enhance immunity. Among all the fractions isolated from O. bracteata, ethyl acetate fraction (Obea) showed good antioxidant activity in Superoxide radical scavenging assay and Lipid peroxidation assay with EC50 value of 95.12 and 80.67 µg/ml, respectively. Silica gel column chromatography of Obea yielded ObD1 fraction which was characterized as Di-isobutyl phthalate (DIBP) using NMR, FTIR and HRMS spectroscopic techniques. DIBP showed antiproliferative activity in human osteosarcoma MG-63, human neuroblastoma IMR-32 and A549 cell lines with GI50 value of 37.53, 56.05 and 47.12 µM, respectively, in MTT assay. In Flow cytometric studies, DIBP has shown disruption of mitochondrial membrane potential (MMP) and enhancement of ROS, indicating the apoptosis induction. The cells were found to be delayed at G0/G1 phase which might be due to the downregulation of Cyclin E and CDK2 as shown in RT-PCR studies. Western blotting analysis revealed an increased expression of p53, caspase 3 and caspase 9 and downregulation of p-NF-kB, p-Akt and Bcl-xl. Molecular docking studies also displayed the interaction of DIBP with p53 (− 151.13 kcal/mol) and CDK1 (− 133.96 kcal/mol). Thus, DIBP has exhibited great potential as chemopreventive/chemotherapeutic agent against osteosarcoma.