PurposeTo evaluate different therapeutic Carnoy's solution formulations on hard human tissues. Materials and methodsAn in vitro study was performed with human teeth (n = 36) and bone fragments (n = 18), randomly divided into two experimental groups (Group I = Carnoy solution with chloroform; Group II = Carnoy solution without chloroform) and a control group (saline solution). The groups were subdivided into pre-conditioning, post-conditioning, and post-conditioning with saline washing. Raman microspectroscopy, Knoop microhardness test, and scanning electron microscopy with energy dispersive X-ray spectroscopy were used. ResultsThere was demineralization of dental structures regarding mineral/matrix and carbonate/phosphate ratios (GI versus GII, p < 0.05). The presence of chloroform resulted in a statistically significant reduction of the teeth surface microhardness (p = 0.036), but not exceeding 0.01 μm. Both GI and GII showed significant structural changes by using scanning electron microscopy with energy dispersive X-ray spectroscopy. ConclusionCarnoy's solution altered the organic and inorganic matrix of the human calcified specimens analyzed in vitro, and its effect was more pronounced when chloroform was present.