Background: Sequential chemical application for irrigating a root canal during chemomechanical debridement can affect the dentin microstructure. Understanding the effects of various irrigants on chemical properties of dentin can elucidate their effects on physical properties and thereby explain the higher incidence of structural failure in endodontically treated teeth. This in vitro research aimed to compare and evaluate the effects of three different irrigating solutions on the chemical structure of root canal dentin in extracted human teeth. Methods: Forty-eight extracted single-rooted mandibular premolar teeth were sectioned at the cemento−enamel junction by a diamond disc and were then randomly assigned to four groups of twelve samples each. The groups were irrigated using 5.25% NaOCl, ozonated olive oil, silver citrate, or distilled water. Dentin sections measuring 1.5 mm were obtained from the root portion and each section and were analyzed using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and electron-dispersive spectroscopy (EDS). FTIR and EDS values are reported as means ± standard deviations. Data were analyzed using an ANOVA and a post hoc Bonferroni test (p < 0.05). Results: A comparison of the FTIR and EDS values among the groups using ANOVA revealed statistically significant differences in the organic and inorganic peak values among the groups. An intergroup comparison between NaOCl with silver citrate and ozonated olive oil revealed significant reductions in the carbonate and phosphate peak values in the NaOCl group (p < 0.05). The EDS values tabulated for the carbon, oxygen, phosphorous, and calcium peak levels showed significant differences between the groups using an ANOVA. An SEM analysis was conducted under 1500× magnification, which revealed smear layer removal in the silver citrate group. Conclusions: The silver citrate solution and the ozonated olive oil caused less changes in the organic and mineral contents of dentin than sodium hypochlorite.