Objective <p>Histone deacetylase (HDAC) inhibitors have emerged as promising cancer therapeutics by regulating gene expression, halting cell cycle progression, and inducing apoptosis. This study explores the structure–activity relationship of 2-mercaptoquinazolin-4(3H)-one derivatives as potential anticancer agents and HDAC inhibitors.</p> Methods <p>The library compounds were prepared via a three-step pathway by incorporating 2-mercaptoquinazoline and a hydroxamic acid moiety. The cytotoxicity of 27 synthesized hydroxamic acid derivatives was evaluated against SW620 (colon cancer), MDA-MB-231 (breast cancer), and MRC-5 (normal lung fibroblast) cell lines. Molecular docking studies on HDAC-isoforms for the <b>4a–i</b> were also performed to identify the essential structural features that contribute to the biological activities.</p> Results <p>The results demonstrated that substituents at the <i>N</i>-3 position significantly influenced anticancer activity, with methyl-substituted derivatives (<b>4a-i</b>) exhibiting the highest cytotoxicity, followed by phenyl-substituted (<b>7a-i</b>) and benzyl-substituted (<b>10a-i</b>) compounds. Among the tested compounds, <b>4a</b> (-H) and <b>4c</b> (7-CH₃) showed as the most potent active compounds, with IC₅₀ values of 4.24 ± 1.16&#xa0;µM and 3.61 ± 0.32&#xa0;µM against SW620 cells, and 2.93 ± 0.68&#xa0;µM and 3.34 ± 0.32&#xa0;µM against MDA-MB-231 cells, respectively. HDAC inhibition assays revealed that <b>4a-d</b> and <b>4&#xa0;g</b> exhibited superior inhibitory activity compared to SAHA. Further investigation of <b>4a</b> and <b>4c</b> in SW620 cells showed that both compounds induced G2/M phase cell cycle arrest and promoted apoptosis, supporting their potential as promising HDAC inhibitors with anticancer properties.</p> Conclusions <p>Among the most active compounds, <b>4a</b> and <b>4c</b> may serve as promising leads for the development of novel HDAC-targeted anticancer therapies.</p>