This paper presents a study on the technique and materials used in the panel painting entitled Pentecostes, attributed to the artist Fernão Gomes (XVIIth century), housed in the Museu Nacional de Arte Antiga, in Lisbon. The study also includes the diagnosis of the conservation state of the painting and the description of the cleaning intervention. For identification of the materials and determination of the conservation state, several analytical techniques were used: micro-FTIR and GC-MS, for identification of the binder and varnish; micro-Raman and micro-FTIR, for identification of fillers and pigments; micro-XRF and SEM-EDX, for elemental analysis; optical microscopy, with visible and fluorescent radiation, and infrared photography, for morphological observation and study of pictorial technique. The analytical approach led to the identification of materials common at the time: gypsum with animal glue, lead white, yellow and red ochres, black earths (based on Mn, Bi, Fe), azurite, possible green earth, lead red, oil and oily varnish with residues of cholesterol. After various cleaning tests, a gel suggested by the literature was chosen (recipe no. 105.4 developed by R. Wolbers) and proved to be effective in the removal of the aged and polymerized varnish.