The use of analytical techniques in cultural heritage research represents an amazing multidisciplinary collaboration. In heritage studies, it is important to pay attention to authenticating, dating, and determining the origin of works of art. Three watercolors were analyzed: Paisaje con árbol, Paisaje con casa campesina and El puente, belonging to the collection of the Central Bank of Costa Rica and attributed to the national artist Fausto Pacheco Hernández. Employing multispectral imaging and innovative computational tools, we determined the state of conservation and biodeterioration of the works evaluated. By using spectroscopic techniques such as Fiber Optics Reflectance (FORS), X–Ray Fluorescense (XRF) and Scanning Electron Microscope–Energy Dispersive X–Ray (SEM–EDX) we identified and compared different pigments in the artworks. Also, we determined the creative process of the artist. Based on our results, Paisaje con árbol was the most likely, of the three paintings, to be attributed to Costa Rican artist Fausto Pacheco, followed by Paisaje con casa campesina and lastly El puente. Finally, we studied the antibacterial and antifungal properties of two of the pigments identified in the paintings (ultramarine blue and yellow ochre). Both the bacterial and fungal isolates were more susceptible to the latter. Applying these kind of studies help to increase the database on the Costa Rican artist and his historical period, providing a better understanding of creative techniques and palettes, in order to strengthen the database for future works.