Pollution of rivers by nitrate is a major issue. Many land use units are considered as net nitrate producers when the input dominates the uptake (e.g. agricultural areas), or in the opposite, net consumers (e.g. wetlands), but the role of their spatial organization and temporal dynamics together across the watershed is unclear. Here, we used a Nitrate-related Ecological Functions (NEF) concept, together with an expert-based analysis in a Geographical Information System, to investigate the role of two opposite landscape types in the nitrate regulation across the Garonne river watershed (France). At any point in a watershed, there is nitrate production (NP) and nitrate removal (NR). The nitrate net balance (NNB) between NP and NR functions can be neutral (NB, Neutral Balance) when nitrate fluxes balance over space and time. The first landscape type, called Actual, was obtained using a set of 7 actual environmental variables, as land cover types, soil organic matter content and wetlands presence. The second landscape type, called Natural, described a non-anthropized landscape, using the same layer types as the Actual landscape. Potentials in NP and NR for each class in each map layer were rated by a set of experts according to their scientific knowledge. NP, NR and by difference, NNB maps were obtained, overlaid and compared to provide an evaluation of the potential for each landscape. In both landscapes, NNB were largely balanced (Actual = 48% and Natural = 67%). In the Actual landscape, NNB were secondly dominated by an imbalance toward NP (43%) and in the Natural landscape secondly imbalanced toward NR (32%). We constructed ‘maps of disagreement’ between both landscapes to provide a spatially explicit assessment of NNB evolution caused by changing land cover. We found that 67% of the agricultural areas and 60% of the artificial areas of the watershed had been subjected to a loss of nitrate ecological functions from Natural to Actual landscapes. Some management practices able to modify these factors may improve ecological functions and diminish the NEF disagreement of the watershed.