The purpose of this observer-blind, randomised, five-cell crossover study was to examine the antibacterial efficacy of an enamel matrix protein derivative (EMD) on established supragingival plaque in vivo. Saline (NaCl) served as a negative control solution and chlorhexidine (CHX) as a positive one. Additionally, the propylene glycol alginate (PGA) vehicle and the 24% ethylenediaminetetra-acetate (EDTA) gel were tested. After professional oral prophylaxis, 14 volunteers refrained from all mechanical oral hygiene measures for the following 48 h to build up plaque. In randomised order, the following procedures were applied: (a) 10 ml of CHX (0.2%) or (b) 10 ml of NaCl were used as a mouthrinse for 1 min each. In the cases of (c) EMD (Emdogain), (d) PGA, or (e) 24% EDTA (PrefGel), 1 ml of each were applied with a syringe on the teeth. Two hours after application, plaque samples were taken from one upper and one lower molar, and the vitality of the biofilm microbiota was examined using the vital fluorescence technique. Biofilm vitality (VF%) was lower for EMD, PGA, and CHX by 19% ( P<0.0001), 22% ( P=0.001), and 35% ( P<0.0001), respectively, than in negative controls. The EDTA showed similar vitality values to NaCl and was therefore not able to affect the biofilm flora significantly. The EMD and PGA displayed significantly reduced biofilm vitality compared to negative controls, which, however, could not reach the effect of the positive control (0.2% CHX). The present results demonstrate for the first time a direct influence of EMD on the vitality of supragingival dental plaque in vivo.