Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are important agents of infectious diarrhoea in humans. Together with the murine-restricted pathogen Citrobacter rodentium, EPEC and EHEC are attaching and effacing (A/E) pathogens that cause hallmark A/E lesions upon colonisation. Their pathogenesis is underpinned by the injection of effector proteins into the host cytosol via the type III secretion system (T3SS), orchestrating subversion of several eukaryotic pathways. However, the individual roles of many effector proteins are not fully understood. Among these are the NleG family of effectors proteins, which harbour a conserved C-terminus that structurally resembles a eukaryotic E3 ubiquitin ligase domain. C. rodentium has three NleG homologs: NleG1, NleG7 and NleG8, which are translocated into eukaryotic cells via the T3SS as demonstrated herein. Biochemical investigations confirmed that NleG1 and NleG8 exhibit E3 ligase activity, reliant on a conserved proline residue P163/P177 that mediates the interaction between E3 and E2 enzymes. As bacteria lack a ubiquitin system, the NleGs are thought to exploit the host ubiquitin code, a notion supported by our findings that NleG7 and NleG8 cause re-localisation of ubiquitin in cultured cells. Both NleG7 and NleG8 bind ubiquitin by yeast two-hybrid assay and the enzymatic activity of NleG8 is required for cytotoxicity in a yeast model. In vivo, single ∆nleG and double ∆nleG7 ∆nleG8 gene deletions did not affect C. rodentium colonisation of the murine gut, but a triple nleG deletion mutant demonstrated attenuated colonisation, suggesting that NleGs contribute to C. rodentium virulence. Reduced faecal Lipocalin 2, an antimicrobial peptide and marker of gut inflammation, was also observed. Significant efforts to find NleG substrates by yeast two-hybrid and mass spectrometry-based screens using transgenic cell lines are outlined, and this work provides a strong foundation for further studies into NleG biology to give insight into A/E pathogenesis.