Transitional waters are characterized by spatial and temporal environmental gradients that make it difficult to discern between anthropogenic and natural stressors on ecosystem functioning. Here, we explored a combination of sampling surveys, analytical techniques focusing on fluorescent dissolved organic matter (FDOM) and remote-sensed products to evaluate the extension and impact of anthropogenic discharges into a Mediterranean coastal lagoon (Mesologgi, Greece). Surface water samples were collected from 20 to 25 sampling points covering the entire surface area of the lagoon during four sampling campaigns. Additional water samples were collected from the adjacent coastal ocean and the main freshwater tributaries to the lagoon, including two Pumping Stations (PS) draining an intensively farmed area, and the emissary of a Wastewater Treatment Plant (WWTP). Remote-sensed products validated the ability of our sampling strategy to capture the temporal and spatial variability of biogeochemical properties within the lagoon. Results showed the PSs and WWTP to be significant sources of nutrients to the lagoon. Parallel factor analysis identified one tryptophan-like, one microbial humic-like and three terrestrial humic-like FDOM components. Principal component analysis identified humic-like FDOM components as potential tracers of anthropogenic inputs into the lagoon. The distributions of the tryptophan-like FDOM component and remote-sensed chlorophyll a were strongly correlated and appeared associated with the location and dispersal of local anthropogenic stressors. Our results showed that the joint analysis of FDOM composition and remote-sensed biogeochemical properties permit to track anthropogenic inputs into complex aquatic environments inclusive of transitional waters, elucidating their impact over the biogeochemistry of the receptor ecosystems.