The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.