This paper reports an unusual green pigment, brochantite (Cu 4 (SO 4 )(OH) 6 ), on 16th-century Portuguese-Flemish paintings, attributed to the Master Frei Carlos workshop. This green mineral is usually identified as an impurity or alteration product in the green pigments verdigris (Cu(CH 3 COO) 2 nCu(OH) 2 ) or malachite (CuCO 3 Cu(OH) 2 ). However, after thorough investigation with a broad range of analytical techniques, it became clear that, in this case, brochantite was applied as a pigment. The abundance, pigment granulometry, and pigment morphology suggest intentional use by this Portuguese-Flemish Master as a natural pigment rather than its accidental use as an alteration product. This seems to be a distinguishable feature to other painters (Flemish and Portuguese) working in Portugal at the beginning of the 16th century.The multi-analytical study of these easel paintings was first performed by physical imaging techniques and material characterization was carried out by optical microscopy, micro-Fourier-transform infrared-spectroscopy, micro-Raman spectroscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry, and micro-X-ray diffraction analysis (XRD).