Background: Studies suggest that decreased microbial diversity due to chemotherapeutic and antibiotic exposure may be associated with acute graft vs host disease (aGvHD) and mortality in patients undergoing allogeneic HSCT. In addition, disruption of the microbiome by antibiotics may lead to intestinal domination by pro-inflammatory bacteria, resulting in increased risk of aGvHD. This relationship has been described in settings with prophylactic antibiotic use, a standard of care in most transplant centers. Here we assessed how the microbiome and GvHD outcomes differ when prophylactic use of antibiotics is avoided. Methods: We collaborated on an observational study (COLLECT) to evaluate changes in microbial diversity over time in subjects undergoing allogeneic HSCT. According to protocol at the University Hospital of Cologne, antibiotics were administered only as empiric treatment for febrile neutropenia or as targeted treatment. Stool was collected weekly from 65 subjects at baseline (pre-HSCT) to day 28 with additional time points taken at day 56, day 90, day 365, and upon diagnosis of intestinal GvHD (GvHD-day 0 and GvHD-day 7). Patients were monitored for incidence of GvHD, including acute GvHD of the liver, intestine, and skin. Microbiome 16SV4 profiles were generated from 381 stool samples. Linear effects models were developed to evaluate the association between Shannon diversity, intestinal domination, and the incidence of intestinal GvHD and mortality. Results: Of the 65 subjects, 28 subjects (42%) went on to develop intestinal GvHD, and 16 subjects (25%) did not survive to day 365. A decline in Shannon diversity was observed during the neutropenic period following HSCT. Subjects who went on to develop intestinal GvHD had significantly lower Shannon diversity at the time of stem cell engraftment (p < 0.0468). Furthermore, lower diversity was observed throughout the study period in subjects experiencing intestinal GvHD. We developed a linear model evaluating the association between mortality and Shannon diversity and found a significant relationship at days 28 and 90 post HSCT (p < 0.0001 and 0.0121, resp). Intestinal domination by Enterobacteriaceae or Enterococcus was significantly associated with the incidence of intestinal GvHD (p < 0.0082) or mortality (p < 0.001), respectively. Conclusions: Data from this observational study (COLLECT) suggests decreases in microbial diversity over time occur in subjects undergoing allogeneic HSCT despite the lack of prophylactic antibiotics. Investigation of whether administration of microbiome therapeutic drugs prior to transplant and/or at the time of engraftment can reduce morbidity and mortality in this high-risk patient population is warranted. Clinical trial information: NCT03148197