A wide variety of cultural heritage materials are susceptible to fungal deterioration. The paper, canvas, and stone constituents of our cultural heritage are subjected to harmful physical and chemical processes as they are slowly consumed by fungi. Remediation of fungal contamination can be costly and risk further damage to cultural artifacts. Early detection of fungal growth would permit the use of relatively noninvasive treatments to remediate fungal contamination before visible or lasting damage to the object has occurred. Current methods used for the detection and measurement of microbial biomass, such as colony counts, microscopic biovolume estimation, and ergosterol analysis are expensive and time consuming, or are inappropriate for use with fungi. Beta-N-acetylhexosaminidase (3.2.1.52) activity provides a reliable estimation of fungal biomass in soil and on building materials. Adapted for use on cultural heritage materials' fluorogenic 4-methylumbelliferyl (MUF) labeled substrate N-acetyl-beta-D-glucosaminide (NAG) was used to detect beta-N-acetylhexosaminidase activity in the fungus Aspergillus niger. Fluorescence increased linearly with fungal biomass and the sensitivity of the assay was comparable to other biochemical techniques. The fluorometric assay was used to monitor fungal biomass on a variety of cultural heritage materials non-destructively, and without the introduction of chemicals or solvents to the surfaces.