Abstract Background This study aimed to investigate clinicopathological and molecular tumour features associated with intratumoral pks⁺Escherichia coli (pks⁺E.coli⁺), pks⁺E.coli^- (non-E.coli bacteria harbouring the pks island), Enterotoxigenic Bacteroides fragilis (ETBF) and Fusobacterium nucleatum (F. nucleatum). Methods We screened 1697 tumour-derived DNA samples from the Australasian Colorectal Cancer Family Registry, Melbourne Collaborative Cohort Study and the ANGELS study using targeted PCR. Results Pks⁺E.coli⁺ was associated with male sex (P < 0.01) and APC:c.835-8 A > G somatic mutation (P = 0.03). The association between pks⁺E.coli⁺ and APC:c.835-8 A > G was specific to early-onset CRCs (diagnosed<45years, P = 0.02). The APC:c.835-A > G was not associated with pks⁺E.coli^- (P = 0.36). F. nucleatum was associated with DNA mismatch repair deficiency (MMRd), BRAF:c.1799T>A p.V600E mutation, CpG island methylator phenotype, proximal tumour location, and high levels of tumour infiltrating lymphocytes (Ps < 0.01). In the stratified analysis by MMRd subgroups, F. nucleatum was associated with Lynch syndrome, MLH1 methylated and double MMR somatic mutated MMRd subgroups (Ps < 0.01). Conclusion Intratumoral pks⁺E.coli⁺ but not pks⁺E.coli^- are associated with CRCs harbouring the APC:c.835-8 A > G somatic mutation, suggesting that this mutation is specifically related to DNA damage from colibactin-producing E.coli exposures. F. nucleatum was associated with both hereditary and sporadic MMRd subtypes, suggesting the MMRd tumour microenvironment is important for F. nucleatum colonisation irrespective of its cause.