Clostridioides difficile infection (CDI) is a major cause of infectious diarrhea among allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. The relationship between CDI and acute graft-versus-host disease (aGVHD) has been a topic of interest, as these 2 conditions may influence each other. We studied the temporal relationship of CDI to aGVHD in the first 100 days post-transplantation in a large cohort of allo-HSCT recipients. We performed a retrospective cohort study of adult patients undergoing their first allo-HSCT at our tertiary care medical center between January 1, 2010, and December 31, 2016. Patients were followed for CDI diagnosis, development of aGVHD, and vital status up to day +100 post-transplantation. Descriptive statistics and multivariate Cox models with CDI as a time-varying covariate and aGVHD and high-grade aGVHD as outcomes were used for data analyses. A total of 656 allo-HSCT recipients were included in the analysis. Of these, 419 (64%) developed aGVHD, and 111 (17%) were diagnosed with CDI within the first 100 days post-transplantation. CDI developed before the onset of aGVHD in 72 of the 84 allo-HSCT recipients (85%) with both CDI and aGVHD. Fidaxomicin was used in the treatment of 57 of the 111 CDI cases (50%), whereas vancomycin was used in 52 (47%). Most of the CDI cases (88%) were diagnosed in the peritransplantation period (between day -10 and day +10). The median time to the development of CDI and aGVHD was 3.5 days (range, -7 to 95 days) and 33 days (range, 9 to 98 days) post-transplantation, respectively. Using multivariate Cox model, the following predictors were significantly associated with the development of aGVHD: CDI (adjusted hazard ratio [aHR], 1.52; 95% confidence interval [CI], 1.17 to 1.97; P = .0018), transplantation from a matched related donor (MRD) compared with a matched unrelated donor (aHR, 0.68; 95% CI, 0.54 to 0.85; P = .0003), and myeloablative versus nonmyeloablative conditioning (aHR, 2.45; 95% CI, 1.80 to 3.34; P < .0001), adjusting for age, sex, race, underlying disease, cytomegalovirus CMV serostatus, transplant source, and receipt of antithymocyte globulin (ATG). There was no association between CDI and high-grade aGVHD after adjustment for age, underlying disease, transplant type, intensity of conditioning, and receipt of ATG (aHR, 1.59; 95% CI, 0.95 to 2.66; P = .0755). CDI after allo-HSCT is associated with increased risk of GVHD when no CDI prophylaxis was used. Further studies examining CDI preventive measures, including prophylaxis, as well as the preservation or reconstitution of the gastrointestinal microbiome in the setting of HSCT are warranted.