Background: Early-onset colorectal cancer (CRC) is a growing public health problem with an incidence rate increase of 7.9% in individuals aged 20-29, 4.9% in those aged 30-39, and 1.6% in those aged 40-49 during 2004-2016. The etiology of this rise is not well established, but there is emerging evidence that the intra-tumoral microbiome may be implicated. We sought to categorize the interaction between diet and intra-tumoral microbiome in early- vs. late-onset CRC. Methods: Intra-tumoral microbiome data were collected from patients enrolled in a retrospective/prospective study of early ( < 45 years) vs. late-onset (≥ 65 years) CRC. Formalin-fixed paraffin-embedded tumor samples were microdissected for tumor vs. non-tumor, then DNA was extracted and analyzed using 16S ribosomal gene sequencing. The DHQ3 diet questionnaire was used to ascertain diet history for a period of 12 months prior to diagnosis. Twelve bacteria of interest were selected for analysis based on increased prevalence and known potential association with CRC. Diet groups of interest for analysis included high-risk groups: fried foods, red meat, and alcohol. Results: A subset of 3 bacteria were further analyzed, Bacillus sp., Fusobacteriumnucleatum, and Moraxella osloensis, based on relation to the group of patients diagnosed with CRC before the age of 45 (N = 8) and after age 65 (N = 12). Bacteria of interest had a similar prevalence in early- (38%, 3/8) and late-onset patients (33%, 4/12). Fifteen of the 20 patients had one or more of the bacteria of interest present. 71% (5/7) of both early- and late-onset patients who had Bacillus present had no alcohol in their diets. 100% (4/4) of the late onset patients with Bacillus present all had the same diet pattern: no tree nuts, high amounts of potato chips, no alcohol, and no red meat. 83% (5/6) of the early onset patients with 1 or more bacteria present had a high amount of potato chips in their diets. 80% (4/5) patients who had none of the bacteria of interest present also had no red meat in their diet. All early- and late-onset patients with Moraxella had a diet that consisted of more high-risk food groups, in particular potato chips. Conclusions: There were no significant differences in intra-tumoral microbiome in patients with early- vs. late-onset CRC. However, there may be an association between certain bacteria and high-risk diets. Results were limited by a small sample size, and further research should be done to better understand the relationship between intra-tumoral microbiome and diet.