Pseudogymnoascus destructans colonizes the wing membrane of hibernating bats with the potential to form dense fungal hyphae aggregates within cupping erosions. These fungal cupping erosions emit a characteristic fluorescent orange-yellow color when the wing membrane is transilluminated with 385 nm ultraviolet (UV) light. The purpose of this study was to create and validate the R package, countcolors, for quantifying the distinct orange–yellow UV fluorescence in bat-wing membrane lesions caused by P. destructans. Validation of countcolors was completed by first quantifying the percent area of 20, 2.5 cm2 images. These generated images were of two known pixel colors ranging from 0% to 100% of the pixels. The countcolors package accurately measured the known proportion of a given color in each image. Next, 40, 2.5 cm2 sections of UV transilluminated photographs of little brown bat (Myotis lucifugus) wings were given to a single evaluator. The area of fluorescence was both manually measured and calculated using image analysis software and quantified with countcolors. There was good agreement between the two methods (Pearson's correlation=0.915); however, the manual use of imaging software showed a consistent negative bias. Reproducibility of the analysis methods was tested by providing the same images to naive evaluators who previously never used the software; no significant difference (P=0.099) was found among evaluators. Using the R package countcolors takes less time than does manually measuring the fluorescence in image analysis software, and our results showed that countcolors can improve the accuracy when quantifying the area of P. destructans infection in bat wing-membranes.