The objective of this study was to develop a DNA probe hybridization procedure using hydrophobic-grid membrane filters (HGMFs) for the detection of E. coli isolates carrying specific virulence or antimicrobial resistance genes. Genes were detected using digoxigenin-labelled probes and an alkaline-phosphatase substrate. Specifically, the eft, estB, and faeG genes encoding the enterotoxins LT, STb, and fimbrial adhesin F4 were targeted. The hybridization procedure was optimized using E. coli strain ECL 7805 which possesses the three targeted genes, and rectal swabs from weaned pigs excreting E. coli positive for these genes, as demonstrated by enrichment PCR.