The aim of this study was to determine the antibacterial effectiveness of either chlorhexidine or calcium hydroxide integrated in gutta-percha points compared with chlorhexidine or calcium hydroxide delivered as gel or paste, respectively. A total of 70 initially sterile roots with open accesses were carried for 1 week in the oral cavities of two volunteers. The roots were then removed, and samples were taken from the root canals for microbial analysis. The roots were medicated with calcium hydroxide paste, 5% chlorhexidine gel, or a chlorhexidine- or calcium hydroxide-containing gutta-percha point. The accesses were closed with bonding material, and the roots incubated for 1 week. After removal of the antimicrobial agents, roots were again checked for bacterial growth. One thioglycolate-soaked paper point was then introduced into each canal, and roots were incubated for 1 week more to observe bacterial regrowth. After 1 week of medication, the absolute bacterial count revealed significant differences compared with the controls. However, only the chlorhexidine-gel and the calcium hydroxide paste group showed no microbial colonization in a considerable number of samples after 1 and 2 weeks.