This article presents an examination of the influence of groundwater flow pattern and denitrification on nitrate migration in the regional recharge zone. For the investigation, both multicomponent chemical tracers and isotopic methods were used. The study revealed different denitrification intensities in regions with groundwater extraction and regions with natural gradients manifested by different levels of potable water contamination by nitrate. A contaminant plume was discovered in shallow parts of the aquifer which percolated into deeper parts of the flow system in the regions with a downward gradient induced by groundwater withdrawal, where the influence of denitrification was limited. The local conditions leading to intense of denitrification, i.e., local changes in geological conditions (low-permeability silt inserts), were also documented. The presented research proves that vertical changes in groundwater chemistry should be examined for effective groundwater resource management and protection, as they are extremely important in regional recharge zones with a downward gradient.