Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a formidable pathogen that uses a type-III secretion system to inject bacterial ‘effector’ proteins directly into host cells. Most effectors that are encoded within the locus of enterocyte effacement (LEE) have been studied extensively. This study aimed to characterise a selection of recently discovered non-LEE-encoded effectors using a variety of model systems. Firstly, a β-lactamase translocation assay was used to demonstrate translocation of novel effectors into host cells. The localisation of selected effectors was then investigated using mammalian cells and a yeast cell model. The effector EspM2 was shown to induce the formation of actin stress fibres in transfected HeLa cells and caused growth retardation when expressed in yeast. A number of NleG effectors also caused growth retardation and morphological changes when expressed in yeast. Growth retardation caused by the effector NleG8-2 was shown to be dependent on three conserved cysteine, aspartic acid and histidine residues. Transcriptomics and a high copy yeast gene suppression screen revealed that NleG8-2 may disrupt yeast physiology by affecting the secretory pathway. This study confirms that the effector repertoire of EHEC O157:H7 is much larger than previously imagined and provides insight into the function of selected novel effectors.