Antibiotic-resistant Enterococcus faecalis and Clostridium difficile are responsible for nosocomial infections in humans, in which they inhabit the same niche. Here, we demonstrate transfer of the conjugative transposon Tn5397 from C. difficile 630 to E. faecalis JH2-2, the first reported gene transfer between these two bacteria. Furthermore, transfer from the E. faecalis EF20A transconjugant to the epidemic ribotype 027 C. difficile strain R20291 was also demonstrated. Tn5397 was shown to use a single specific target site in E. faecalis; it also has specific target sites in C. difficile. These experiments highlight the importance of continual monitoring for emerging resistances in these bacteria.