This is a technical study of the extant murals of the 17th Capilla de la Virgen del Rosario located within Iglesia San José, San Juan, Puerto Rico. The primary objectives of this investigation were to: document existing mural campaigns, establish a chronology of mural painting through analysis of materials and techniques, evaluate the conditions of the paintings and to determine possible deterioration mechanisms, and propose recommendations for their conservation and interpretation. In-situ documentation including color digital photography, extensive field notes, and mapping of visible painting campaigns were conducted. This was followed by a materials analysis of select campaigns’ substrate, binders, and pigments. Test methods included gravimetric analysis and XRD of substrate plasters, examination of cross-sections and pigment dispersions, EDS analysis of pigments, and FTIR analysis of binders. The results of this study found six distinct mural campaigns and established a chronology which attributed painting phases to the Dominican, Jesuit, and Vincention orders of the Catholic Church. Notable iconography include the 17th century mer creatures (la serena), and the mid-19th century depiction of the Battle of Lepanto. Substrate analysis revealed a lean plaster mix in the enfoscado as an intrinsic cause of failure, further aggravated by continued water infiltration. Water ingress has created an environment supporting threatening deterioration mechanisms including abundant chloride salts, and biological growth contributing to failing paint layers and plasters. The Rosario Chapel murals are highly significant and warrant a comprehensive strategy for their conservation and interpretation through a collaborative process involving all stakeholders.