Introduction The purpose of this in vitro study was to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite (NaOCl) and Chlorhexidine (CHX), and relative molecular weight of the components. Methods Using commercially available chlorhexidine gluconate (CHXg), a 2% solution was formed and mixed in a 1:1 ratio with commercially available NaOCl producing a brown precipitate. The precipitate as well as a mixture of precipitate and pure chlorhexidine diacetate (CHXa) was then analyzed using 1D and 2D NMR spectroscopy. Results The 1D and 2D NMR spectra were fully assigned, in terms of chemical shifts of all proton and carbon atoms in intact CHX. This permitted identification of CHX breakdown products with and without the aliphatic linker present, including lower molecular weight components of CHX that contained a para-substituted benzene that was not para-chloroaniline (PCA). Conclusions Based on this in vitro study, the precipitate formed by NaOCl and CHX is composed of at least two separate molecules, all of which are smaller in size than CHX. Along with native CHX, the precipitate contains two chemical fragments derived from CHX, neither of which are PCA.