Objectives: The first aim of this project is to establish a mixed species biofilm containing Enterococcus faecalis on dentine in vitro. The second aim is to determine if E. faecalis has an increased resistance to sodium hypochlorite when grown as a mixed species biofilm compared with a single species biofilm. Method: Biofilms were established in a flow cell chamber following confirmation from pilot studies. One flow cell was inoculated with E. faecalis only. Another flow cell was inoculated with Fusobacterium nucleatum and allowed to establish for 48 hours followed by an inoculum containing E. faecalis and Streptococcus sanguinis. Biofilms were left to grow for 4 weeks and were then treated with 0.9% sodium hypochlorite followed by 5% sodium thiosulphate for 1 minute each. Physiological saline acted as a control. Biofilms were harvested by sonication and cellular viability of E. faecalis was determined by serial plating onto selective media. Qualitative analyses were carried out on images of control and treated discs taken by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Results: After 4 weeks a single and mixed species biofilm was established on dentine discs which were confluent, dense and adherent. There were both live and dead bacteria present within the biofilms. Four out of eight treated discs in the mixed species biofilm displayed positive bacterial counts compared to only two discs in the single species biofilm. The remaining discs showed patent dentinal tubules and levels of bacteria which were non detectable. Conclusion: A mixed species biofilm containing E. faecalis was successfully grown on dentine in vitro. Results showed a trend indicating E. faecalis is more resistant to sodium hypochlorite when grown as a mixed species biofilm.