Based on the rational design of DNA intercalators and Topo-II inhibitors and taking into consideration the main pharmacophoric features of doxorubicin (Dox) as a reference standard, we theoretically designed novel substituted tetrahydropyrimidine analogues (T). The designed analogues (T) were investigated for their inhibitory potential towards the hybrid DNA and Topo-II target receptor using molecular docking. Interestingly, the theoretically designed analogue T with a 3,4,5-trimethoxy phenyl side chain was found to be the superior candidate, achieving a binding score of -7.06 kcal mol, compared with two reference standards, doxorubicin (Dox) and a co-crystal ligand (EVP). Moreover, the docked candidates (T, Dox, and EVP) were further subjected to molecular dynamics simulations for 500 ns. Furthermore, MM-GBSA calculations showed that the target candidate (T) achieved superior Δ binding energy (-33.86 kcal mol) compared with Dox and EVP. Moreover, T was found to be the most promising candidate that could be conveniently synthesized based on its order in the chemical synthesis scheme. In addition, to evaluate the antiproliferative activity and scope of compound T, we requested the National Cancer Institute (NCI) to test it against nine cancer cell types. Interestingly, compound T exhibited very strong antiproliferative activity with a mean GI% of 122% and a mean GI of 4.10 μM. It exhibited the highest anticancer activity towards all 59 cell lines. Moreover, the binding interaction of compound T with calf thymus DNA (ctDNA) was examined using various techniques, such as spectrofluorimetry, UV-vis spectrophotometry, viscosity measurements, ionic strength measurements, and thermodynamics to confirm its mechanism of action. Investigating the intermolecular binding interaction between small compounds and DNA can provide valuable insights for designing drugs with enhanced effectiveness and improved targeted activities.