Enterotoxigenic Escherichia coli (ETEC), being the major cause of post-weaning diarrhoea (PWD) in newly weaned piglets, induces poor performance and economic losses in pig production. This functional in vitro screening study investigated probiotic strains for use in suckling piglets as a prophylactic strategy towards PWD. Nine strains were evaluated based on their ability to: enhance intestinal epithelial barrier function, reduce adherence of ETEC F18 to intestinal cells, inhibit growth of ETEC F18, and grow on porcine milk oligosaccharides. Strains included in the screening were of the species Lactobacillus, Enterococcus, Bifidobacterium and Bacillus. Our in vitro screening demonstrated genus-, species and strain-specific differences in the mode of action of the tested probiotic strains. Some of the tested bifidobacteria were able to grow on the two porcine milk oligosaccharides, 3'-sialyllactose sodium salt (3'SL) and Lacto-N-neotetraose (LNnT), whereas most lactic acid bacteria strains and both Bacillus subtilis strains failed to do so. All probiotic strains inhibited growth of ETEC F18 on agar plates. All but the bifidobacteria reduced binding of ETEC F18 to Caco-2 cell monolayers, with the Enterococcus faecium strain having the most profound effect. All three lactic acid bacteria and Bifidobacterium animalis subsp. lactis counteracted the ETEC F18-induced permeability across Caco-2 cell monolayers with the E. faecium strain exhibiting the most pronounced protective effect. The findings from this in vitro screening study indicate that, when selecting probiotic strains for suckling piglets as a prophylactic strategy towards PWD, it would be advantageous to choose a multi-species product including strains with different modes of action in order to increase the likelihood of achieving beneficial effects in vivo.