Enterohaemorrhagic E. coli (EHEC) are well described food-borne and zoonotic pathogens. Ruminants, particularly cattle, are considered the principle reservoir host. Although O157:H7 is the archetypal strain for this E. coli pathotype, several other pathogenic serogroups are recognised, including O26, O111 and O103, particularly in Australia. Although operational hygiene during slaughter and processing of meat is important to control and prevention of EHEC transmission to humans, reduction of pathogen carriage in the reservoir host offers the optimal approach to control. Apart from reducing EHEC burdens at the time of slaughter, reservoir control can potentially reduce environmental pathogen contamination and direct zoonotic transmission. Of several approaches explored for reducing bovine carriage of EHEC, vaccination has thus far indicated the most promise. Products currently available target E. coli O157:H7, are designed for use primarily North American beef production systems, and have other limitations that reduce their application more broadly. The goal of the current study was to screen novel E. coli antigens for efficacy in reducing bovine carriage of non-O157 EHEC serotypes. Using a combination of bioinformatic interrogation of EHEC strains and in vitro phenotypic methods, a cluster of antigens that have potential EHEC O26 colonisation blocking effects were identified. These antigens were tested by parental immunisation of experimental cattle that were orally inoculated with EHEC O26 and observing EHEC O26 recto-anal junction (RAJ) colonisation rates for three weeks post-challenge. Intimin, a recognised effector protein for E. coli O157:H7 colonisation and pathogenesis, significantly reduced bovine RAJ colonisation by EHEC O26. Two other novel antigens were similarly associated with EHEC O26 anti-colonisation effects. Use of these antigens in a combinatorial vaccine, particularly if combined with targeted delivery systems, may offer a method for reduction of bovine-human transmission risk for non-O157 EHEC.