This study aimed to evaluate the characterization of chemical interaction of root canal irrigants on the surface of EndoSequence root repair materials using spectroscopy analysis. Round discs of putty and paste were obtained and immersed in saline, sodium hypochlorite (NaOCl), or chlorhexidine. On the surface of chlorhexidine treated putty, diffuse red pigmentation was detected by Raman analysis and diffuse black pigmentation having unusual needle-like shaped crystals was detected by scanning electron microscopy. This pigmentation formed from nitrogen-rich compounds detected by Raman, infrared spectroscopy, and X-ray diffraction analysis. Mineral/ amide ratios were increased by NaOCl and significantly decreased by chlorhexidine. Carbonate/phosphate ratios showed no significant changes by NaOCl, while a significant decrease by chlorhexidine. A full half of maximum width Raman of the phosphate band was significantly increased by NaOCl, while decreased by chlorhexidine. In conclusion; nitrogen-rich compounds produced by chlorhexidine altered the surface morphology of the putty without crystallinity reduction. However, NaOCl reduced the organic fillers that affected its binding to phosphate ions. This reaction affects the chemical properties and perfection of root repair material. The clinicians should be aware of different irrigants to be used after repairing the perforation.