Enterococci are opportunistic pathogens that are frequently a source of nosocomial infections and it is their resistance to antibiotics and their ability to form biofilms that represent important virulence traits. Normally, in healthy individuals it is a harmless commensal that is usually found in the intestine. This thesis firstly studies signal peptidases (SPases), which play an essential role in protein translocation. Interestingly, E. faecium was found to contain three type I SPases. Many proteins that are secreted are virulence factors, and the aim was to delete one or more of the SPases and study the effect of its removal on virulence. Unfortunately no mutants were obtained suggesting that the genes were essential. To establish if the genes were essential an inducible integration vector was constructed, but due to time constraints this could not be tested further. Biofilm formation was studied in both E. faecium and E. faecalis. The presence of the Enterococcal Surface Protein (Esp) in E. faecium was shown to increase hydrophobicity, and therefore also increase biofilm formation. Similarly, E. faecalis isolates that were good biofilm formers were also more hydrophobic in nature. The expression of Esp in E. faecium was studied under different conditions; these studies indicated that the highest level of Esp expression was found in biofilms cells. This growth-dependent manner Esp expression was not observed in E. faecalis BS12297. Surprisingly, Esp in E. faecium was also shown to have a role in ampicillin resistance, which was identified using calorimetry. This method proved to be a sensitive and rapid method to analyse antibiotic resistance. In the gut, bacteria encounter various adverse conditions, such as low pH and the presence of bile salts. Here we investigated the effects of bile salts on biofilm formation in E. faecium and E. faecalis and demonstrated that biofilm formation is induced at physiological concentrations of bile salts. In E. faecium the presence of bile salts caused an increase in initial attachment, microcolony formation and EPS production. Various factors were investigated, including hydrophobicity, cell growth, cell morphology, Esp expression and the production of extracellular polymeric substance (EPS). In E. faecium, only EPS production appeared to play a role, but the stimulation of biofilm formation due to bile salts is still to be fully explained.