In the Pra Basin of Ghana, groundwater is increasingly becoming the alternative water supply due to the continual pollution of surface water resources through illegal mining and indiscriminate waste discharges into rivers. However, our understanding of hydrogeology and the dynamics of groundwater quality remains inadequate, posing challenges for sustainable water resource management. This study aims to characterize groundwater recharge by determining its origin and mechanism of recharge prior to entering the saturated zone and to provide spatial estimates of groundwater recharge using stable isotopes and water level measurements relevant to groundwater management in the basin. Ninety (90) water samples (surface water and groundwater) were collected to determine stable isotope ratios of oxygen (δ18O) and hydrogen (δ2H) and chloride concentration. In addition, ten boreholes were installed with automatic divers to collect time series data on groundwater levels for the 2022 water year. The Chloride Mass Balance (CMB) and the Water Table Fluctuation (WTF) methods were employed to estimate the total amount and spatial distribution of groundwater recharge for the basin. Analysis of the stable isotope data shows that the surface water samples in the Pra Basin have oxygen (δ18O) and hydrogen (δ2H) isotope ratios ranging from −2.8 to 2.2‰ vrs V-SMOW for δ18O and from −9.4 to 12.8‰ vrs V-SMOW for δ2H, with a mean of −0.9‰ vrs V-SMOW and 0.5‰ vrs V-SMOW, respectively. Measures in groundwater ranges from −3.0 to −1.5‰ vrs V-SMOW for δ18O and from −10.4 to −2.4‰ vrs V-SMOW for δ2H, with a mean of −2.3 and −7.0‰ vrs V-SMOW, respectively. The water in the Pra Basin originates from meteoric source. Groundwater has a relatively depleted isotopic signature compared to surface water due to the short residence time of infiltration within the extinction depth of evaporation in the vadose zone. Estimated evaporative losses in the catchment range from 51 to 77%, with a mean of 62% for surface water and from 55 to 61% with a mean of 57% for groundwater, respectively. Analysis of the stable isotope data and water level measurements suggests a potential hydraulic connection between surface water and groundwater. This hypothesis is supported by the fact that the isotopes of groundwater have comparatively lower values than surface water. Furthermore, the observation that the groundwater level remains constant in months with lower rainfall further supports this conclusion. The estimated annual groundwater recharge in the catchment ranges from 9 to 667 mm (average 165 mm) and accounts for 0.6% to 33.5% (average 10.7%) of mean annual precipitation. The total estimated mean recharge for the study catchment is 228 M m3, higher than the estimated total surface water use for the entire Pra Basin of 144 M m3 for 2010, indicating vast groundwater potential. Overall, our study provides a novel insight into the recharge mechanism and spatial quantification of groundwater recharge, which can be used to constrain groundwater flow and hydrogeochemical evolution models, which are crucial for effective groundwater management within the framework of the Pra Basin’s Integrated Water Resources Management Plan.