Oral mucositis (OM) is a debilitating multifactorial complication of hematopoietic stem cell transplantation (HSCT). We hypothesized that the oral microbiome is disturbed during transplantation and may partially account for the variability in OM severity across patients. In this single-center study, we prospectively collected saliva samples weekly in a cohort of 184 adults undergoing allogeneic HSCT. A total of 625 samples were collected starting from 7 days before HSCT to 34 days post-transplant. Samples underwent 16S rRNA gene sequencing. Sixty pre- and post-HSCT samples underwent mass spectrometry-based metabolomic profiling (Metabolon, Durham, NC). OM was graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Myeloablative conditioning regimens were used in 70% of patients. The median time to OM development was 7 days; 58% and 43% of patients developed grade 2-4 and grade 3-4 OM, respectively. Pre-transplantation oral α-diversity, as measured by the Shannon Index, was similar to that of healthy controls (n=20) (p=0.460), but later decreased, reaching a nadir on day 12 (Fig. A). Among patients with grade 2-4 OM, the reduction in α-diversity over time was more pronounced compared to transplant-recipients having no or only mild OM (grade 0-1) (Fig. B). In the group treated with MTX (n=135), patients harboring Rothia, Kingella, and Atopobium in the saliva before stem cells infusion were more likely to develop grade 3-4 OM than their counterparts. Furthermore, in samples collected between days 7 to 13, there was a higher abundance of Methylobacterium among patients with grade 3-4 OM, while Treponema and the genus TG5 (Fig. 3) were more common in patients with grade 0-1 OM (Fig. C). Finally, metabolic profiling of saliva collected pre and post HSCT showed marked changes before and after transplantation among patients who developed mucositis. Microbiome-associated metabolites enriched in patients with mucositis included histidine, phenylalanine, tyrosine, and tryptophan. In this largest analysis of the oral microbiome in HSCT recipients, we demonstrate that the oral microbiota is disrupted during allogeneic HSCT. Microbiota injury is more profound in patients who developed oral mucositis. Our findings indicate that there may be a distinct microbiological and metabolic signature in patients with mucositis, with some changes preceding the clinical phenotype. These may serve as potential intervention points to treat and prevent mucositis.