Onosma species have been used commonly for traditional medicine for years due to their bioactive compounds content. Onosma bourgaei aerial part was extracted with hexane and methanol successively. The methanol extract was subjected to chromatographic techniques to isolate allantoin (1), 3,4-dihydroxybenzaldehyde (2), luteolin-7-O-glucoside (3), apigenin-7-O-beta-glucoside (4), diosmetin-7-O-beta-glucoside (5), rosmarinic acid (6), and globoidnan A (7). The structure of isolated compounds were identified by spectroscopic techniques such as 1D-NMR, 2D-NMR, FTIR, and LC-TOF/MS/MS. Antiproliferative activity of extract and natural compounds were carried out using HeLa (human epithelial cervix adenocarcinoma, ATCC (R) CCL-2 (TM)), HT29 (human colorectal adenocarcinoma, ATCC (R) HTB38 (TM)), MCF7 (human mammary gland adenocarcinoma, ATCC (R) HTB22 (TM)), and A549 (human lung carcinoma, ATCC (R) CCL185 (TM)) cancerous cells and normal cells, FL (human epithelial amnion cell, ATCC (R) CCL62 (TM)). Lactate dehydrogenase (LDH) was performed for cytotoxicity. The compounds, 4, 6, and 7 displayed the strong antiproliferative activity against corresponding cell lines. Apigenin-7-O-beta-glucoside (4) revealed the excellent activity on HeLa, HT29, A549, and MCF6 cancer cell lines with the values of (IC50, mu M) 167.3, 196.8 181.1, and 203.5, respectively, compared standard compound, cisplatin.