EnteroaggregativeEscherichia coli(EAEC) and enterohemorrhagicE. coli(EHEC) areE. colipathotypes associated with unmanageable diarrhea in children and adults. An alternative to the treatment of infections caused by these microorganisms is the use of the bacteria of theLactobacillusgenus; however, the beneficial effects on the intestinal mucosa are specific to the strain and species. The interest of this study consisted of analyzing the coaggregation properties ofLactobacillus caseiIMAU60214, as well as the effect of cell-free supernatant (CSF) on growth and anti-cytotoxic activity in a cell model of the human intestinal epithelium for an agar diffusion assay (HT-29) and the inhibition of biofilm formation on plates of DEC strains of the EAEC and EHEC pathotypes. The results showed thatL. caseiIMAU60214 exhibits time-dependent coaggregation (35–40%) against EAEC and EHEC that is similar to the controlE. coliATCC 25922. The CSF showed antimicrobial activity (20–80%) against EAEC and EHEC depending on the concentration. In addition, the formation and dispersion of biofilms of the same strains decrease, and the proteolytic pre-treatment with catalase and/or proteinase K (1 mg/mL) of CSF reduces the antimicrobial effect. When evaluating the effect in HT-29 cells pre-treated with CFS on the toxic activity induced by the EAEC and EHEC strains, a reduction of between 30 and 40% was observed. The results show thatL. caseiIMAU60214 and its CSF have properties that interfere with some properties associated with the virulence of the EAEC and EHEC strains that cause intestinal infection, which supports their use for the control and prevention of infections caused by these bacteria.