The remarkable potential of probiotics in preventing and treating various illnesses has captured the attention of researchers and health enthusiasts alike. However, amidst the enthusiasm, it is crucial to understand the specific effects of each probiotic strain. It is evident that probiotics exhibit immunomodulatory effects, enhance the functionality of the gut barrier, and mitigate inflammation. However, it becomes apparent that a deep comprehension of the unique mechanisms exhibited by individual probiotic strains is imperative for optimizing their therapeutic efficacy. Potential risks are associated with probiotic use, raising the need for careful consideration when employing these interventions. Probiogenomics, which involves high-throughput techniques, can help reveal uncharacterized strains and allow for the rational selection of new probiotics. This study evaluates the potential of the commensal Escherichia coli CEC15 strain as a probiotic through in silico, in vitro, and in vivo analyses, compared to the E. coli Nissle 1917 reference strain. The strains were submitted to genomic analysis in search of traits with potential beneficial activity and to evaluate the safety of each strain. Analyses of genomic islands, bacteriocins production, antibiotic resistance, homeostasis proteins, and proteins with potential adhesive properties were performed, followed by in vitro studies to evaluate GIT passage survival and adhesion of the strain to the intestinal wall. To assess the safety in a complex organism, BALB/c mice were fed daily, for 13 days, with 10 10 CFU of either strain, and the clinical signs, microscopic analysis of the intestinal architecture, intestinal permeability, and microbiota modulation were evaluated. In parallel, the strains' immunomodulatory, anti-inflammatory, and protective effects were also assessed in a murine model of 5-FU-induced mucositis. The findings demonstrate that both strains exhibit potential for immunomodulation, reinforcement of the intestinal barrier, anti-inflammatory effects, and modulation of the intestinal microbiota. In silico results for CEC15 showed fewer traits related to pathogenicity than in Nissle 1917, with fewer genes associated with toxins, especially the absence of colibactin production gene cluster, a potent genotoxic agent. Besides antibiotic-resistance genes, most genes found did not present the phenotype in vitro nor were associated with transposable elements. Furthermore, the presence of stress tolerance proteins and adhesion-related proteins was high, so the CEC15 strain presented a better digestion survival rate and higher levels of adhesion to intestinal cells. CEC15 also positively affected the host and its intestinal microbiota in both healthy animals and against 5FU-induced intestinal mucositis. CEC15 strain holds promise as a probiotic for modulating the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings could have implications for treating gastrointestinal disorders, particularly inflammatory bowel disease. However, further research is essential to assess the safety and effectiveness of the CEC15 strain in humans.