Preliminary data have suggested that taurolidine may bear promising disinfectant properties for the therapy of bacterial infections. However, at present, the potential antibacterial effect of taurolidine on the supragingival plaque biofilm is unknown. To evaluate the antibacterial effect of taurolidine on the supragingival plaque biofilm using the vital fluorescence technique and to compare it with the effect of NaCl and chlorhexidine (CHX), 18 subjects had to refrain from all mechanical and chemical hygiene measures for 24 h. A voluminous supragingival plaque sample was taken from the buccal surfaces of the lower molars and wiped on an objective slide. The sample was then divided into three equal parts and mounted with one of the three test or control preparations (a) NaCl, (b) taurolidine 2% and (c) CHX 0.2%. After a reaction time of 2 min, the test solutions were sucked of. Subsequently, the plaque biofilm was stained with fluorescence dye and vitality of the plaque flora was evaluated under the fluorescence microscope (VF%). Plaque samples treated with NaCl showed a mean VF of 82.42 ± 6.04%. Taurolidine affected mean VF with 47.57 ± 16.60% significantly (p < 0.001, paired t test). The positive control CHX showed the lowest mean VF values (34.41 ± 14.79%; p < 0.001 compared to NaCl, p = 0.017 compared to taurolidine). Taurolidine possesses a significant antibacterial effect on the supragingival plaque biofilm which was, however, not as pronounced as that of CHX.