Probiotics are microorganisms that can promote beneficial results to the host when administered in adequate quantities. Escherichia coli Nissle 1917 (EcN) is the most commonly known Gram-negative probiotic and its beneficial activities have been demonstrated in many studies since its discovery in the 20th century. We recently isolated from newborn rats’ feces a primo colonizer E. coli strain, called CEC15, with promising probiotic activities. It can colonize the gut of germ-free mice without modifying inflammatory parameters and displays substantial anti-inflammatory effects in a DSS-induced colitis model. In this work, we tested the potential probiotic activity of CEC15 in an in vivo model of intestinal mucositis induced by 5-fluorouracil (5-FU) administration. Bacteria were administered to BALB/c mice (male, 5 weeks old) daily in a 1.1010 CFU/animal dosage. EcN was administered as probiotic activity control. After 10-days of administration, the mucositis was induced by intraperitoneal injection of 5-FU (300mg/Kg), and the animals were sacrificed 72h later. Treatment was kept after mucositis induction. CEC15 promoted reduction of inflammatory histological scores, intestinal permeability, weight loss, and neutrophilic infiltrate, while EcN was only able to prevent an increase in intestinal permeability. An In vitro study of bacterial survival in the gastrointestinal tract (INFOGEST protocol) shows that both strains resist digestion conditions reaching a survival rate of 50% and 10% for CEC15 and EcN, respectively, in an initial dosage of 1.109 CFU/mL. Both strains also exhibit resistance to high concentrations of bile salts. Genomic analysis shows CEC15 belonging to the B1 phylogroup while EcN belongs to the B2 group. CEC15 genome contains 12 putative pathogenic islands against 25 on EcN and does not have the pks island, which encodes for the production of colibactin in EcN, a compound that has been associated with its genotoxicity side effect. Besides being necessary for more assays to confirm the safety and immunomodulation of the CEC15 strain, as well as the effects of its metabolites and extracellular vesicles produced, these results show a promising future for this strain for probiotic applications.