Display omitted •A multi-analytical approach based on four different analytical methods was used for the first time.•The approach is very effective in characterizing organic materials used in the past.•The ointment bases of historical ointment remains from the 18th century were identified.•Contemporary pharmacists often modified the formulations to suit their needs. A multi-analytical approach based on four different analytical methods was used for the first time to study six historical ointments from the 18th century belonging to the baroque pharmacy of Capuchin Monastery in Hradčany (Prague, Czech Republic) in order to gain information on the ointment formulation, the presence of active substances, and also possible chemical modification deriving from the procedures used for their preparation. All ointments were initially characterized by gas chromatography/mass spectrometry following saponification, extraction and derivatization to identify the main classes of ingredients. The volatile organic compounds emitted by the ointments were then characterized by gas chromatographic analysis following solid-phase microextraction, providing complementary information on the possible more volatile and active ingredients present in the ointments. Finally, the botanical/animal origin of triacylglycerols was investigated by liquid chromatography-high resolution mass spectrometry, while the presence of beeswax was proved by flow injection analysis. The results obtained were used to hypothesize the probable original medical purpose of the ointments by comparing the results with historical recipes and the period literature. The use of this comprehensive multi-analytical approach allowed us to contribute to the knowledge of ancient ointments and to obtain information on the alteration of the chemical compounds in the ointment formulations.