Background Clostridioides difficile (CD) is the most common healthcare-associated enteric infection. There is currently limited epidemiological evidence on CD incidence in South Africa. Aim To estimate the burden of CD infection (CDI) in the South African public sector between 1 July 2016 and 30 June 2017. Methods A retrospective cohort study utilizing secondary data was conducted to describe the epidemiology of CD in South Africa. We assessed the patient-level association between variables of interest, CD, and CD recurrence, by undertaking both univariate and multivariable analysis. Adjusted incidence rate ratios (aIRR) were calculated utilizing multivariable Poisson regression. The incidence of CD, CD recurrence and CD testing was estimated by Poisson regression for various levels of care and provinces. Results A total of 14 023 samples were tested for CD during the study period. After applying exclusion criteria, we were left with a sample of 10 053 of which 1 860 (18.50%) tested CD positive. A positive and significant association between CDI and level of care is found, with patients treated in specialized tuberculosis (TB) hospitals having a five-fold increased adjusted incidence risk ratio (aIRR) for CDI (aIRR 4.96 CI95% 4.08–6.04,) compared to those managed in primary care. Patients receiving care at a secondary, tertiary, or central hospital had 35%, 66% and 41% increased adjusted incidence of CDI compared to those managed in primary care, respectively. National incidence of CDI is estimated at 53.89 cases per 100 000 hospitalizations (CI95% 51.58–56.29), the incidence of recurrence at 21.39 (CI95% 15.06–29.48) cases per 1 000 cases and a recurrence rate of 2.14% (CI95% 1.51–2.94). Conclusion Compared to European countries, we found a comparable incidence of CD. However, our estimates are lower than those for the United States. Compared to high-income countries, this study found a comparatively lower CD recurrence.