Cancer is considered a global health burden and its occurrence has increased over the last century. Therefore, one of the main concerns of medicinal chemists is to discover novel therapeutic candidates for cancer treatment. In the present work, two novel series of quinazolinone derivatives have been synthesized and biologically investigated as anticancer agents. In the first series, thirty-one compounds of quinazolinone-chalcone hybrids were designed, synthesized, and investigated for in vitro cytotoxic activity against five human cancer cell lines namely, epidermoid carcinoma A431, lung adenocarcinoma A549, fibrosarcoma HT-1080, mammary adenocarcinoma MDA-MB-231, and prostate adenocarcinoma PC-3, as well as, normal AG01523 cells using the MTT assay. Most of the new hybrids showed remarkable cytotoxic activity in the low micromolar range. Compounds 14, 16, 22, 27, 33, 34, and 35 had shown the highest cytotoxicity among the tested compounds against the epidermoid carcinoma A431 with IC50 ranging from 1 to 2 μM. Notably, the tested compounds displayed no or little cytotoxicity against the normal human skin fibroblasts. Flow cytometric cell cycle analysis performed on A431 cells revealed that the most cytotoxic compounds arrested A431 cells in the S- or G2/M- phase or both and induced a marked increase in the percentage of subdiploid events, indicating the stimulation of cell death by apoptosis. A further mechanistic study demonstrated that quinazolinone-chalcone hybrids induced apoptosis via induction of caspase-3 and PARP-1 cleavage. Moreover, a molecular docking study was performed for the target chalcones to investigate their mode of binding to PARP-1. Finally, a conclusive structure-activity relationship (SAR) study was formulated to facilitate the future design of powerful apoptotic candidates targeting both caspase-3 and PARP-1 proteins. The second series of compounds was designed as quinazolinone-aminothiazole hybrids and most of the compounds in this series displayed modest cytotoxic activity against at least one cell line in the low micro molar range. Structure-activity relationship study revealed that substituents at meta-position of quinazolinone 3- phenyl ring were better for activity than para-substituted derivatives. Bulky substituents such as iodo and bromo substituents at the para position caused marked decrease in cytotoxic activity. However, more compounds are required to be synthesized in order to formulate a conclusive structure-activity relationship in this series. Further mechanistic investigations are required to be carried out in the future. Spectral analysis was used to confirm the structures of the novel target compounds and their purity was checked using elemental analysis.