A series of novel hybrid pyrazolotriazolopyramidine derivatives was designed and synthesized in synthetically useful yields. All the new synthesized compounds were biologically evaluated in vitro for their cytotoxic activities against a panel of three cancer cell lines namely, HepG-2, MCF-7, and HCT-116. The results of cytotoxic evaluation indicated that compounds 12, and 11 exhibited the most prominent cytotoxic effect against all tested cell lines with IC₅₀ values ranging from 12.41 to 22.18 µM comparable to that of doxorubicin as a control drug (IC₅₀ values of 8.17 and 9.27 µM). Moreover, the most potent compound was further evaluated for its topoisomerase II inhibitory activities and DNA intercalating affinities as potential mechanisms for its anti-proliferative activities. In particular, compound 12 exhibited higher intercalative activity with IC₅₀ value of 30 µM than doxorubicin (31 µM). Interestingly, compound 12 displayed a significant topoisomerase II inhibitory activity with IC₅₀ value of 0.055 µM. Furthermore, molecular docking study was also performed in order to understand the binding mode in the active site and explain the anti-cancer results with prospective target.