This work studied the potential of probiotic candidate Limosilactobacillus reuteri E to ameliorate intestinal mucositis in vitro. By PathogenFinder analysis of genome sequence, the strain was not predicted to be pathogenic. Genes responsible for adhesion (MapA, EF-Tu) and immunomodulation (e.g., EpsD, EpsF, EpsC, enzymes responsible for the synthesis of exopolysaccharides) abilities were localized in the strain genome. In the co-cultivation model of Caco-2 cells and L. reuteri E, lactobacilli up-regulated gene expression of occludin and claudin 1 (tight junction proteins) and paralemmin (receptor-like molecule) in Caco-2. The prophylactic and therapeutic potential of L. reuteri E to modulate intestinal mucositis was investigated by its administration to Caco-2 cells before and after 5-FU treatment. In bacteria, both prophylactic and therapeutic approaches resulted in significantly higher levels of EpsF glycosyltransferase gene expression (1.5-fold and 3.4-fold, respectively) compared to lactobacilli co-cultivated with untreated Caco-2 cells. In Caco-2 cells, significantly increased gene expression of occludin and claudin 1 in prophylaxis was observed. Moreover, lactobacilli used in prophylaxis ameliorated 5-FU-induced toxicity in Caco-2 cells, as shown by increased cell viability. This may be related to improved anti-inflammatory-to-pro-inflammatory ratio (higher A20 and IL-10 vs. IL-1β and IL-8 gene expression) observed in prophylactic conditions compared to the therapeutic lactobacilli application to Caco-2 cells. The results obtained suggest that L. reuteri E may be more effective in the prophylaxis of 5-FU-induced intestinal mucositis than in therapeutic use.