Cyclophosphamide (Cy) is a prodrug that is mainly bioactivated by the cytochrome enzyme CYP2B6. Several other enzymes are also involved in its bioactivation and affect its kinetics. Previous studies have shown the effect of the enzymes’ genetic polymorphisms on Cy kinetics and its clinical outcome. These results were controversial primarily because of the involvement of several interacting enzymes in the Cy metabolic pathway, which can be also affected by several clinical factors as well as other drugs interactions. In this article, we present the effect of CYP2B6 polymorphisms on Cy kinetics since it is the main bioactivating enzyme, as well as discussing all previously reported enzymes and clinical factors that can alter Cy efficacy. Additionally, we present explanations for key Cy side effects due to its extrahepatic bioactivation. Finally, we discuss the role of busulphan in conditioning regimens in the Cy metabolic pathway as a clinical example of drug-drug interactions involving several enzymes. By the end of this article, our aim is to have provided a conclusive summary of Cy pharmacogenomics and the effect of its kinetics. The use of these findings in new strategies for Cy personalized patient dose adjustment will certainly help in optimizing the Cy dose and in improving the clinical outcome.