Gut microbiota dysbiosis with increased pathogenic bacteria and decreased beneficial bacteria is associated with colorectal cancer (CRC) development. This study examined the effect of a newly developed probiotic formula in modulating CRC-related bacteria. We developed a probiotic formula containing three bifidobacteria (B. adolescentis,B. longum, andB. bifidum) based on the identification of bacterial species that showed significant correlations with CRC-related bacteria includingFusobacterium nucleatum(Fn),Lachnoclostridium sp. m3,Clostridium hathewayi(Ch), andBacteroides clarus(Bc). We co-culturedFnwith each bifidobacterium or the combined formula and examined the growth ofFnby qPCR. The three individual bifidobacteria significantly inhibited the growth ofFncompared to the control treatment (24~65% inhibition; allp< 0.001). The combination of the three bifidobacteria showed a greater inhibitory effect onFngrowth (70% inhibition) than the individual bifidobacteria (allp< 0.05). We further examined the effect of the probiotic formula in a pilot study of 72 subjects (40 on probiotics; 32 with no intervention) for 4 weeks and followed them up for 12 weeks. The relative fecal abundances of the bifidobacteria in the formula and the CRC-related markers (Fn,m3,Ch, andBc) were quantitated by qPCR before and after the intervention, and the combined CRC risk score (4Bac;Fn, m3,Ch, andBc) was evaluated. Subjects with probiotics intervention showed significantly increased abundances of the bifidobacteria from week 2 to week 5 compared to baseline (p< 0.05), and the abundances dropped to baseline levels after the cessation of the intervention. There were significant decreases in the levels of CRC-related markers (Fnandm3) and the CRC risk score (4Bac) from week 2 to week 12 compared to baseline levels (p< 0.05) in the intervention group but not in the control group. A novel probiotic formula containingB. adolescentis,B. longum, andB. bifidumwas effective in inhibiting the growth ofF. nucleatumin vitro and improving the gut microbial environment against CRC development.