Prostate cancer (PC) is the second most common tumor in males worldwide. The lack of effective medication and the development of multidrug resistance towards current chemotherapeutic agents urge the need to discover novel compounds and therapeutic targets for PC. Herein, seven synthesized 2,3-dihydroquinazolin-4(1H)-one analogues were evaluated for their anticancer activity against PC3 and DU145 cancer cell lines using MTT, scratch-wound healing, adhesion and invasion assays. Besides, a liquid chromatography mass spectrometry (LC–MS)-based metabolomics approach was followed to identify the biochemical pathways altered in DU145 cancer cells upon exposure to dihydroquinazolin derivatives. The seven compounds showed sufficient cytotoxicity and significantly suppressed DU145 and PC3 migration after 48 and 72 h. C2 and C5 had the most potent effect with IC50 < 15 µM and significantly inhibited PC cell adhesion and invasion. Metabolomics revealed that C5 disturbed the level of metabolites involved in essential processes for cancer cell proliferation, progression and growth including energy production, redox homeostasis, amino acids and polyamine metabolisms and choline phospholipid metabolism. The data presented herein highlighted the importance of these compounds as potential anticancer agents particularly C5, and pointed to the promising role of metabolomics as a new analytical approach to investigate the antiproliferative activity of synthesized compounds and identify new therapeutic targets.