Background and Objectives: Mycological diagnosis of onychomycosis is based on direct microscopy using external fluorophores to visualize fungal tissue in nail samples and agar culture. Ultraviolet fluorescence excitation imaging (u-FEI) has shown potential in monitoring biological processes by exploiting variations in autofluorescence. This study aimed at assessing the potential of a handheld u-FEI system as a practical screening tool for fungal nail infections. Study Design/Materials and Methods: Ninety samples from 29 patients with microscopy-confirmed fungal infection and 10 control samples from healthy participants were collected (n = 100). Using a prototype u-FEI system (single bandpass 25 mm filter with a central pass wavelength of 340 nm and a bandwidth of 12 nm, 295 nm excitation flash, resolution of 640 × 480), images of all samples were acquired under standardized conditions. Average and maximum fluorescence intensity image values in arbitrary units (AU) of manually delineated regions of interests were quantitated and statistically assessed for significant differences between healthy and mycotic samples. Results: UV-images clearly depicted all 100 nail samples, with a visibly stronger signal in infected samples. Statistically significant differences (P < 0.05) in signal intensity between mycotic samples and healthy controls were observed for maximum and average fluorescence values. Mean fluorescence values of onychomycotic samples showed 23.9% higher maximum (mycotic: 34.9 AU [standard deviation [SD] 4.7]; healthy: 28.2 AU [SD 1.9]) and 10.2% higher average (mycotic: 27.6 AU [SD 2.0]; healthy: 25.0 AU [SD 0.7]) signal intensity values. Receiver operating characteristic curves demonstrated excellent discriminatory ability (area under the curve > 0.9). Analysis of fluorescence measurements of the reference standard demonstrated very low variation (coefficient of variation = 0.62%). Conclusion: Quantitation of u-FEI intensities enables differentiation between healthy and mycotic nail samples, constituting a potential point-of-care tool for cost-effective screening for onychomycosis at a primary care level. Lasers Surg. Med.