Carnoy's solution has been widely undertaken as adjunctive therapy for benign odontogenic tumors. Although its outcomes were highly investigated, little information is available regarding its mechanism of action and the role of chloroform in composition. The aim of this study was to characterize Carnoy's solution (with and without chloroform) in order to survey its physicochemical and pharmacological properties. UV/Vis spectroscopy, Raman microspectroscopy, viscosity assessment, pH analysis, and ion (Fe3+) concentration were studied. All solutions were prepared by the same operator and assayed at each 7 days within a 4‐week period. All solutions depicted acidic pH whereas the viscosity was increased by the addition of chloroform. Starting in the seventh day after preparation, solutions with chloroform showed iron precipitation when nonfiltered. Carnoy's solution without chloroform was stable during the survey period as evidenced by Raman spectroscopy. In conclusion, Carnoy's solution possesses an acidic characteristic, it is stable when stored at room temperature, and precipitates iron salts when in the presence of chloroform in a nonfiltered solution. The chloroform increases the viscosity of the solution and makes its pH slightly more acidic when compared to the Carnoy's solution without chloroform. Raman spectroscopy favored the evaluation of components' stability in solutions by using the spectra analysis and the established correlations between the peaks in different periods.