Fusobacterium nucleatum is an oral bacterium known to colonize colorectal tumors, where it is thought to play an important role in cancer progression. Recent advances in sequencing and phenotyping of F. nucleatum have revealed important differences at the subspecies level, but whether these differences impact the overall tumor ecology, and tumorigenesis itself, remain poorly understood. In this study, we sought to characterize Fusobacteria in the tumor microbiome of a cohort of individuals with CRC through a combination of molecular, spatial, and microbiologic analyses. We assessed for relative abundance of F. nucleatum in tumors compared to paired normal tissue, and correlated abundance with clinical and pathological features. We demonstrate striking enrichment of F. nucleatum and the recently discovered subspecies animalis clade 2 (Fna C2) specifically in colon tumors that have biofilms, highlighting the importance of complex community partnerships in the pathogenesis of this important organism. ### Competing Interest Statement C.L.S. has received research funding to Johns Hopkins University from Bristol-Myers Squibb and Janssen, and royalties from Up to Date outside the submitted work. J.R.W. reports equity ownership of Resphera Biosciences.

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