A series of novel 2,4-diaminopyrimidines containing piperidine and piperazine moieties were synthesized via an efficient one-pot methodology. The bioassay tests demonstrated that compounds 27 and 28 displayed much stronger antitumor activities against four human cancer cell lines (HepG2, A549, MDA-MB-231 and MCF-7) than positive control fluorouracil. Particularly, compound 28 showed a two-fold improvement compared to fluorouracil in inhibiting MDA-MB-231 and A549 cell proliferation with IC50 values of 7.46 and 12.78 μM, respectively. Further flow-activated cell sorting analysis revealed that the most promising compound 28 displayed a significant effect on G2/M cell-cycle arrest in a dose-dependent manner in MDA-MB-231 cells.