Purpose: This laboratory study assessed the influence of surface roughness and contact time on the formation of a multi-species biofilm on dental materials (adhesive patch, composite, amalgam and enamel). Materials and Methods: Rough and smooth specimens of each material were prepared and the mean surface roughness was assessed profilometrically. The biofilms were then allowed to grow either for 15 min or 15 h respectively on salivapreconditioned specimens of each material, and colony-forming units on blood agar were counted (N = 9/group). Surface morphology was assessed using a scanning electron microscope. Results: No difference was found in the biofilm formation rate among all the materials that were tested. After a short incubation period, a statistical significant difference between smooth and rough samples could be detected on amalgam and on the resin composite material (P ≤ 0.001). Conclusions: Surface roughness may influence the initial biofilm adherence, but differences vanish following growth and maturation. The multi-species biofilm offers a reliable laboratory model for studying plaque formation. Keywords: dental materials, multi-species biofilm, SEM, surface roughness