Objectives Evidence has shown that a prophylactic antibiotic regimen of flucloxacillin and gentamicin for orthopaedic surgery was associated with increased rates of post-operative acute kidney injury (AKI). This resulted in changes in the national antibiotic policy recommendation for orthopaedic surgical prophylaxis. This study aimed to assess whether this change from flucloxacillin and gentamicin to co-amoxiclav was associated with changes in the rates of AKI and Clostridium difficile infection (CDI).Methods An observational study and interrupted time series analyses were used to assess rates of post-operative AKI separately in patients undergoing neck of femur (NOF) repair and other orthopaedic operations that required antibiotic prophylaxis. Incidence rate ratios were used to evaluate changes in CDI rates.Results Following the change in policy, from flucloxacillin and gentamicin to co-amoxiclav, there was a relative change in rates of post-operative AKI of −63% (95% CI −77% to −49%) at 18 months in the other orthopaedic operations group. In the NOF repair group, there was no change in the rate of post-operative AKI [−10% (95% CI −35%–15%)] at 18 months. The incident rate ratio for CDI in the other orthopaedic operations group was 0.29 (95% CI 0.09–0.96) and in the NOF repair group was 0.76 (95% CI 0.28–2.08).Conclusions The use of co-amoxiclav for antibiotic prophylaxis in orthopaedic surgery was associated with a decreased rate of post-operative AKI compared with flucloxacillin and gentamicin and was not associated with increased rates of CDI.