New series of triazolo[4,3-c]quinazolines were designed, synthesized and their structures were elucidated using different spectroscopic techniques. They were evaluated for their in vitro antitumor activity against HepG2, MCF-7, PC-3, HCT-116 and HeLa cancer cell lines using MTT assay. It was found that all compounds showed variable in vitro cytotoxicity. Distinct derivatives exhibited higher inhibitory activity against the tested cell lines with IC<sub>50</sub> values ranging from 8.27 to 10.68 µM using DOX standard (IC<sub>50</sub> = 4.17-8.87 µM). In vitro epidermal growth factor receptor (EGFR) inhibition assay was performed. Results revealed that compounds 8, 19 and 21 exhibited worthy EGFR inhibitory activity with IC<sub>50</sub> values ranging from 0.69 to1.8 µM in comparison to the reference drug Gefitinib (IC<sub>50</sub> = 1.74 µM). Further investigation showed that active candidates 8, 19 and 21 caused cell cycle arrest at the G2/M phase, and interestingly, induced cell death by apoptosis of MCF-7 cells cumulatively with 7.14, 17.52 and 24.88%, respectively, compared with DOX as a positive reference (29.09%). Molecular modeling studies, including docking, flexible alignment and surface mapping, were also done to study the interaction mode into the active site of EGFR kinase domain. There was a good agreement between modeling results and biological results. ADMET analysis and parameters of Lipinski's rule of five were calculated. Pharmacokinetic parameters showed that compound 8 had more expected penetration through blood brain barrier than Gefitinib. The present work displayed new triazoloquinazoline based derivatives with potent cytotoxicity and promising EGFR inhibition activity.