: Despite longstanding contributions to oceanography, similar use of fluorescence light detection and ranging (LiDAR) in lake settings is not routine. The potential for ship-mounted, multispectral Ultraviolet Fluorescence LiDAR (UFL) to provide rapid, high-resolution data in variably turbid and productive lake conditions are investigated here through a series of laboratory tank and field measurements carried out on Lake Balaton, Hungary. UFL data, calibrated empirically to a set of coinciding conventionally-analyzed samples, provide simultaneous estimates of three important parameters-chlorophyll a (chl a), total suspended matter (TSM) and colored dissolved organic matter (CDOM). Successful UFL retrievals from both laboratory and field measurements were achieved for chl a (0.01–378 mg· m− 3; R= 0.83–0.92), TSM (0.1–130 g· m− 3; R= 0.90–0.96) and CDOM (0.003–0.125 aCDOM (440); R= 0.80–0.97). Fluorescence emission at 685 nm is shown through tank measurements to display robust but distinct relationships with chl a concentration for the two cultured algae species investigated (cyanobacteria, Cylindrospermopsis raciborskii, and chlorophyta, Scenedesmus armatus). The ratio between fluorescence emissions measured at 650 nm, related to the phycocyanin fluorescence maximum, to that at 685 nm is demonstrated to effectively distinguish these two species. Validation through both laboratory measurements and field measurements confirmed that site specific calibration is necessary. This study presents the first known assessment and application of ship-mounted fluorescence LiDAR in freshwater lake conditions and demonstrates the use of UFL in measuring important water quality parameters despite the more complicated hydro-optic conditions of inland waters.