The ability of probiotics to adhere to the intestinal epithelium likely plays an important role in their colonization of the gastrointestinal tract. Here, we performed high-throughput screening (HTS) for suitable characteristics of potential probiotic bacteria using attachment and colonization ability through a C. elegans surrogate in vivo model. A total of 100 strains of lactic acid bacteria (LAB) isolated from infant feces were subjected to the colonization assay using C. elegans intestine. Based on colonization ability, we showed that nine isolates have a high attachment ability during whole experimental periods (up to 168 h), compared to Lactobacillus rhamnosus strain GG as a control. Also, through the use of an in vitro cell attachment model, nine isolates revealed highly binding activity to the mucus layer. Next, the selected 9 isolates were assayed for their survival ability when exposed to acidic and bile conditions as well as cholesterol reduction and the utilization of prebiotic substrates. As a result, the isolated nine strains were determined to be highly resistant to acid and bile conditions. In addition, they have significant activity for the reduction of cholesterol and utilization of several prebiotic substrates as a carbon source. Finally, the selected nine strains were identified by either L. rhamnosus or L. plantarum (4 strains for L. rhamnosus and 5 strains for L. plantarum, respectively). Taken together, we propose that the direct colonization of probiotics using C. elegans may be applicable to the rapid screening of valuable probiotic strains in vivo.