Enterotoxigenic Escherichia coli (ETEC) are a major cause of sporadic diarrhea disease in humans, affecting mainly infants in developing countries and travelers from industrialized countries visiting tropical or subtropical areas. In this study, we screen the antagonistic activity by inoculating wells among ETEC agar cultures to assess inhibition zones created by the lactobacilli spent culture supernatant (SCS) from healthy infant stool. Only three isolates possessed antagonistic activity, acid and bile tolerance and could adhere to the cultured human intestinal C2BBel (Caco-2) cell line. Isolate identification using API 50CHL strips showed that they belonged to different Lactobacillus species, i.e., Lactobacillus acidophilus RY2, Lactobacillus salivarius MM1 and Lactobacillus paracasei En4. The SCS still had an inhibitory effect on ETEC after heating (100 degrees C, 15 min). The lactate dehydrogenase treatment or the pH of SCS was adjusted to neutral (pH 7.2) to reduce the SCS inhibitory effect. Antimicrobial activity was performed by incubating the lactic acid bacteria (LAB)-SCS with ETEC suspension. After 4h of co-culture, ETEC growth was inhibited. This study suggests that L. acidophilus RY2, L. salivarius MM1 and L. paracasei En4 could be used as an effective control for ETEC.