The aim of the present study was to compare the antibacterial effect of calcium hydroxide (CH), triantibiotic mixture (TAM), and CH in combination with chlorhexidine (CHX), sodium hypochlorite (NaOCl) or colchicine (COL) against Enterococcus faecalis (E. faecalis) in surface and deep dentinal tubules. Seventy five fresh single-rooted human teeth were infected and divided into five experimental groups (n=15). The experimental groups were treated with CH+distilled water, CH+CHX, CH+NaOCl, CH+COL+distilled water and TAM+distilled water. Dentin chips obtained from surface and deep dentin of these root canals were prepared and analyzed by counting the number of colony forming units. There was significant difference between groups in the surface dentin (p<0.05). TAM showed higher antibacterial activity compared to CH-containing groups. There was no significant difference between TAM and CH-containing groups in the deep dentin (p>0.05). CH-containing medications and TAM can be used as effective disinfectants in treatment of infected root canals.