AbstractNanotechnology-based strategies can overcome the limitations of conventional cancer therapies. Hence, novel series of pyrimidine Schiff bases (4–9) were employed in the synthesis of selenium nanoparticle forms (4NPs–9NPs). All selenium nano-sized forms exerted greater inhibitions than normal-sized compounds, far exceeding 5-fluorouracil activity. Compound4showed effective anti-proliferative activity against MCF-7(IC503.14 ± 0.04 µM), HepG-2(IC501.07 ± 0.03 µM), and A549(IC501.53 ± 0.01 µM) cell lines, while its selenium nanoform4NPsshowed excellent inhibitory effects, with efficacy increased by 96.52%, 96.45%, and 93.86%, respectively. Additionally,4NPsoutperformed4in selectivity against the Vero cell line by 4.5-fold. Furthermore,4NPsexhibited strong inhibition of CDK1(IC500.47 ± 0.3 µM) and tubulin polymerase(IC500.61 ± 0.04 µM), outperforming4and being comparable to roscovitine (IC500.27 ± 0.03 µM) and combretastatin-A4(IC500.25 ± 0.01 µM), respectively. Moreover, both4and4NPsarrested the cell cycle at G0/G1 phase and significantly forced the cells towards apoptosis. Molecular docking demonstrated that4and4NPswere able to inhibit CDK1 and tubulin polymerase binding sites.