Introduction: This study aimed to present the synthesis and characterization of four novel analogs of cyclophosphamide (2, 3, 4, 7) and their related precursors (1, 5, 6) and assess their anticancer activity against breast cancerous (MCF-7) and normal (HUVEC) cells., Method: Notably, 2-(bis(2-chloroethyl)amino)-1,3,2-diazaphospholidine 2-oxide ((2)) and 2-(bis(2-hydroxyethyl)amino)-1,3,2-diazaphospholidine 2-oxide ((7)) exhibited concentration- dependent cytotoxicity against the MCF-7 cell line, with IC50 values of 8.98 and 28.74 μM, respectively., Result: Annexin V/PI staining and ROS assays demonstrated reduced cell viability and mitochondrial dysfunction. in silico studies involving DFT-D optimization and Molegro virtual docking against B-DNA dodecamer and STAT3 receptors revealed enhanced interactions for certain compounds compared to cyclophosphamide., Conclusion: Importantly, the in silico and in vitro results corroborated each other, supporting the potential anticancer efficacy of these novel analogs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)