Antibiotics have been empirically used for human inflammatory bowel disease, being limited to short periods. Probiotics are able to attenuate intestinal inflammation due to its immunomodulatory properties, being considered as safe when chronically administered. The aim was to test the association of minocycline, a tetracycline with immunomodulatory properties, and the probiotic Escherichia coli Nissle 1917 (EcN) in a mouse model of reactivated colitis. For this purpose, female C57BL/6J mice were assigned to different groups: non-colitic and dextran sodium sulfate (DSS)-control groups (without treatment), minocycline (50 mg/kg/day; p.o.), EcN (5 × 108 CFU/day; p.o.), and minocycline plus EcN treated groups. Colitis was induced by adding DSS in the drinking water (3%) for 5 days; 2 weeks later, colitis was reactivated by subsequent exposure to DSS. The inflammatory status was evaluated daily by a disease activity index (DAI); colonic damage was assessed histologically and biochemically by evaluating mRNA relative expression of different mediators by qPCR. Finally, a microbiological analysis of the colonic contents was performed. Minocycline and EcN exerted intestinal anti-inflammatory effect and attenuated the reactivation of the colitis, as shown by the reduced DAI values, being these effects greater when combining both treatments. This was evidenced histologically and biochemically, by reduced expression of TNFα, IL-1β, IL-2, MIP-2, MCP-1, ICAM-1, iNOS and MMP-9, together with increased MUC-3 and ZO-1 expression. Finally, the altered microbiota composition of colitic mice was partially restored after the different treatments. In conclusion, EcN supplementation to minocycline treatment improves the recovery of the intestinal damage and prevents the reactivation of experimental colitis.