Objectives: This study aims to evaluate and compare radiological, biomechanical,histopathological,histomorphometricandimmunohistochemical effects of povidone iodine (PVP-I), hydrogen peroxide (HPO) and chlorhexidine gluconate (CHG) on fracture healing in their minimum cytotoxic and most efficient concentrations.Materialsandmethods:This experimental animal study, conducted between April 2018 and January 2019, included 48 male Sprague Dawley® rats (aging 9 weeks; weighing 356 g) which were randomly divided into four groups: control (saline), HPO, PVP-I and CHG. Rat model of femoral fracture was established and intramedullary fixation was applied. Solutions were applied to fracture region in determined concentration and time, and all subjects were sacrificed on Day 28. Extracted femurs were investigated radiologically by micro-computed tomography. Then, all groups were divided into two random groups to be evaluated biomechanically, histopathologically, histomorphometrically and immunohistochemically.Results: In histopathological evaluation, inflammation score of CHG group was significantly lower than other groups, and inflammation score of PVP-I group was significantly lower than control and HPO groups (p<0.05). Biomechanically, flexural strength (σbend) (megapascal) values of CHG and control groups showed similar results, but there was no significant difference between all groups (p>0.05). In immunohistochemical localization of bone morphogenic protein (BMP)-4, osteoblast and chondroblast histoscores (H-scores) of HPO group were significantly lower than other groups, and chondroblast H-score in CHG group was lower than control and PVP-I groups (p<0.05). In immunohistochemical localization of BMP-7, osteoblast H-score was significantly higher in CHG group than other groups (p<0.05).Conclusion: We determined that CHG 0.05% solution had no negative effect on the fourth week of fracture healing histopathologically, immunohistochemically and biomechanically, and is an alternative irrigative to normal saline.