The aim of this study was to gain a deeper insight into the properties of egg, used as a binding medium, and its interactions with water and organic solvents. The research focused on egg tempera films prepared in July 2007 and on a tempera layer prepared by researchers of the Smithsonian’s Museum Conservation Institute in 1995. It also included a sixteenth-century panel painting from the Pinacoteca di Siena. Two paint samples from this painting were analyzed by gas chromatography coupled with mass spectrometry and identified egg as the binding medium. Ethanol or acetone, isooctane, and water were used to test for leaching. In general, the main leaching occurs from the fresher samples: using solvents of different polarity (from isooctane to water), the lipid components are the most removed. It also was observed that pigmented layers are less affected by leaching phenomena than a layer without pigments, in particular for lipid components. Finally, it was noticed that unsaturated fatty acids were extracted mainly from fresh samples, with less from the 1995 tempera and nothing from the sixteenth-century painting. However, the study carried out on this painting showed that leaching is more pronounced for the lipid components, and amino acids were also detected. These results were unexpected because mild cleaning tests were believed not to affect a 500-year-old painting. These results might be regarded as guidelines to take into account for cleaning paintings.