The purpose of this study was to evaluate the disinfection of dentinal tubules with diclofenac sodium (DS), N‐acetylcysteine (NAC) and calcium hydroxide (CH). Contaminated dentinal blocks were divided into two control and seven experimental groups (n = 15): CH, DS, NAC, CH + 5% DS, 50% CH + 50% DS, CH + 5% NAC and 50% CH + 50% NAC. After seven days, dentine debris was obtained from two depths of 100 and 200 µm. The bacterial load was assessed by counting the number of colony‐forming units (CFUs). Pure DS exhibited maximum antibacterial activity at both depths. At 200 µm, it showed statistically significant differences with all the other groups (P < 0.05). Mixing CH with either 5% or 50% of DS and NAC did not increase the antibacterial efficacy (P > 0.05). Pure DS was most effective in disinfecting dentinal tubules, and mixing CH with DS or NAC is not recommended.