The aim of the present study was to map the painting materials, degradation processes, and biological features present on the mural painting in the church of St. Mary in Beram (Croatia) to study their possible interaction and produce information helping the preservation of this valuable painting. The research was conducted on micro samples of painting materials taken from different sites along the painting and the characterization of the present fungal species was carried out. The painting samples, together with observable patinas and degradation products, were studied by optical microscopy (OM), scanning electron microscopy, energy-dispersive spectroscopy (SEM/EDS), Fourier-Transform Infrared spectroscopy, and powder X-ray diffraction. Fungal diversity was studied using cultivation methods followed by OM and SEM analyses in addition to molecular analysis. The results contribute to the characterization of the original painting materials, successively added materials and occurred interventions, to the understanding of degradation progressions and fungal biotransformation processes. A mineral, cumengite, a copper-based pigment extremely rarely used in art, was found. Its occurrence together with barium sulfate, gypsum, and calcium oxalate possibly produced by microbiological activity was studied and information was added regarding the composition of painting materials in St. Mary church mural cycle.