Clinical outcomes of tamoxifen (TAM) treatment show wide interindividual variability. Comedications and genetic polymorphisms of enzymes involved in TAM metabolism contributes to this variability. Drug–drug and drug–gene interactions have seldom been studied in African Black populations. We evaluated the effects of commonly co-administered medicines on TAM pharmacokinetics in a cohort of 229 South African Black female patients with hormone-receptor positive breast cancer. We also investigated the pharmacokinetic effects of genetic polymorphism in enzymes involved in TAM metabolism, including the variants CYP2D6*17 and *29, which have been mainly reported in people of African descent. TAM and its major metabolites, N-desmethyltamoxifen (NDM), 4-OH-tamoxifen, and endoxifen (ENDO), were quantified in plasma using the liquid chromatography-mass spectrometry. The GenoPharm open array was used to genotype CYP2D6, CYP3A5, CYP3A4, CYP2B6, CYP2C9, and CYP2C19. Results showed that CYP2D6 diplotype and CYP2D6 phenotype significantly affected endoxifen concentration (P < 0.001 and P < 0.001). CYP2D6*17 and CYP2D6*29 significantly reduced the metabolism of NDM to ENDO. Antiretroviral therapy had a significant effect on NDM levels and the TAM/NDM and NDM/ENDO metabolic ratios but did not result in significant effects on ENDO levels. In conclusion, CYP2D6 polymorphisms affected endoxifen concentration and the variants CYP2D6*17 and CYP2D6*29 significantly contributed to low exposure levels of ENDO. This study also suggests a low risk of drug–drug interaction in patients with breast cancer on TAM.