The CoMiniGut in vitro model mimicking the small intestine of piglets was used to evaluate four probiotic strains for their potential as a preventive measure against development of diarrhea in weaned pigs. In the in vitro system, piglet digesta was inoculated with pathogenic enterotoxigenic Escherichia coli F4 (ETEC F4), and the short-chain fatty acid profile and the gut microbiota composition were assessed. A total of four probiotic strains were evaluated: Enterococcus faecium (CHCC 10669), Lactobacillus rhamnosus (CHCC 11994), Bifidobacterium breve (CHCC 15268) and Faecalibacterium prausnitzii (CHCC 28556). The significant differences observed in metabolite concetration and bacterial enumeration were attributed to variation in inoculating material or pathogen challenge rather than probiotic treatment. Probiotic administration influenced the microbiota composition to a small extend. Learnings from the present study indicate that the experimental setup, including incubation time and choice of inoculating material, should be chosen with care.