This study aimed to evaluate the removal of 2‐hydroxyisocaproic acid (HICA) from the root canal system and its effect on the bond strength of the mineral trioxide aggregate (MTA). 126 single‐rooted teeth were divided into 3 experimental groups (n = 36) that were treated with double antibiotic paste (DAP), HICA and calcium hydroxide (CH) and one control group (n = 18). In the first part, 18 teeth from each experimental group (n = 54) were examined to remove HICA from the root canal. In the second part, 72 teeth (3 experimental groups (n = 54) and one control group (n = 18)) were used for the evaluation of the push‐out bond strength of MTA. There was no statistically significant difference between HICA, DAP and CH residues (p > 0.05). HICA group showed significantly less push‐out bond strength (p < 0.05). DAP, HICA and CH could not be removed entirely from the root canal. HICA significantly reduced the bond strength of the MTA.