Aims: Dicloxacillin is used to treat staphylococcal infections and we have previously shown that dicloxacillin is an inducer of cytochrome P450 enzymes (CYPs). Here, we employed a translational approach to investigate the effect of a treatment with dicloxacillin on warfarin efficacy in Danish registries. Furthermore, we assessed dicloxacillin as an inducer of CYPs in vitro. Methods: We conducted a register‐based study and analysed international normalized ratio (INR) levels in chronic warfarin users before and after short‐ and long‐term use of dicloxacillin (n = 1023) and flucloxacillin (n = 123). Induction of CYPs were investigated in a novel liver model of 3D spheroid primary human hepatocytes at the level of mRNA, and protein and enzyme activity. Results: Short‐ and long‐term dicloxacillin treatments decreased INR levels by −0.65 (95% confidence interval [CI]: −0.57 to −0.74) and −0.76 (95% CI: −0.50 to −1.02), respectively. More than 90% of individuals experienced subtherapeutic INR levels (below 2) after long‐term dicloxacillin treatment. Flucloxacillin decreased INR levels by −0.37 (95% CI: −0.14 to −0.60). In 3D spheroid primary human hepatocytes, the maximal induction of CYP3A4 mRNA, protein and enzyme activity by dicloxacillin were 4.9‐, 2.9‐ and 2.4‐fold, respectively. Dicloxacillin also induced CYP2C9 mRNA by 1.7‐fold. Conclusion: Dicloxacillin induces CYPs and reduces the clinical efficacy of warfarin in patients. This effect is substantially exacerbated during long‐term treatment with dicloxacillin. The in vitro results corroborated this drug–drug interaction and correlated to the clinical findings. Caution is warranted for warfarin patients that initiate dicloxacillin or flucloxacillin, especially for a long‐term treatment of endocarditis. [ABSTRACT FROM AUTHOR]