Chlorhexidine has been incorporated in different varnishes to provide a slow release system on the tooth surface in order to reduce mutans streptococci. To provide an alternative vehicle for chlorhexidine with better adhesion properties compared to resin-based varnishes, glass ionomer cement (GI) has been suggested. However, one disadvantage for glass ionomers is a longer setting time compared to the resin-based varnishes. The aim with this study was to compare the fissure penetration and antibacterial characteristics of a glass-ionomer cement (GI) with a GI containing chlorhexidine gluconate (GI-CHX), and GI-CHX with added tataric acid (GI-CHX-TA) to reduce its setting time. Antibacterial properties against mutans streptococci were assessed by agar diffusion. GI, GI-CHX and GI-CHX-TA were applied with a microbrush on the occlusal surfaces of 4, 4 and 6 extracted molars respectively. After setting of cements, sections were ground with 1 mm intervals and photographed. The fissure penetration and adaptation of the cements were scored excellent, acceptable or unacceptable under blind conditions according to a standard. Seventy percent scored excellent with GI-CHX-TA (n = 54) compared with 40% with GI-CHX (n = 48) and 38% with GI (n = 40), (p < 0.05). GI-CHX and GI-CHX-TA had significant better antibacterial properties compared to GI or GI with added tataric acid only.