Escherichia coli is one of the main pathogens that impacts swine production. Given the need for methods for its control, the in vitro effect of lactic acid bacteria (LAB) and their metabolites against E. coli F4 was evaluated through cell culture and microbiological analysis. The strains Limosilactobacillus fermentum 5.2, Lactiplantibacillus plantarum 6.2, and L. plantarum 7.1 were selected. To evaluate the action of their metabolites, lyophilized cell-free supernatants (CFS) were used. The effect of CFS was evaluated in HT-29 intestinal lineage cells; in inhibiting the growth of the pathogen in agar; and in inhibiting the formation of biofilms. The bioprotective activity of LAB was evaluated via their potential for autoaggregation and coaggregation with E. coli. The CFS did not show cytotoxicity at lower concentrations, except for L. fermentum 5.2 CFS, which is responsible for cell proliferation at doses lower than 10 mg/mL. The CFS were also not able to inhibit the growth of E. coli F4 in agar; however, the CFS of L. plantarum 7.1 resulted in a significant decrease in biofilm formation at a dose of 40 mg/mL. Regarding LAB, their direct use showed great potential for autoaggregation and coaggregation in vitro, thus suggesting possible effectiveness in animal organisms, preventing E. coli fixation and proliferation. New in vitro tests are needed to evaluate lower doses of CFS to control biofilms and confirm the bioprotective potential of LAB, and in vivo tests to assess the effect of LAB and their metabolites interacting with animal physiology.