Although root canal instruments remove most of the content from the main root canal space, disinfection or irrigation plays an indispensable role in all areas of the root canal system, especially in parts that are inaccessible by instruments. The originality of this study was to investigate the effect of two novel disinfection techniques on the surface hardness of resin-based endodontic sealers using atomic force microscopy (AFM). Forty extracted single-rooted maxillary central human teeth were prepared and divided into four groups according to treatment methods. The first group was irrigated with saline and served as a control, other groups irrigated with sodium hypochlorite (NaClO); gaseous ozone; and light-activated disinfection (LAD). The groups were divided into two subgroups, according to the obturation method used: subgroup A: gutta-percha and AH plus; and subgroup B: EndoREZ/resin-coated cones. After obturation, atomic force microscopy (AFM) measurement was performed to analyze the surface hardness of the sealers. There was a significant difference between group 1A and group 3A (p<0.05). Group 3B had the highest surface hardness values that were statistically different (p<0.05). When disregarding the sealers, the ozone possessed statistically higher surface hardness values than the other groups in all root thirds (p<0.05). The use of ozone and LAD may alter the surface hardness of resin-based sealers. The use of AFM can be considered an alternative hardness test techonology for sealing material. SCANNING 38:141-147, 2016. (c) 2015 Wiley Periodicals, Inc.