In the last four decades, the Southern Amazon (south of 8°S) has shown changes in the spatial and temporal patterns of its hydro-climatic components, leading to drier conditions. Due to climate and land-use changes, this region is considered as a zone under biophysical transition processes. Previous studies have documented a complex interaction between climate and deforestation either on a large-scale or based on limited in situ data, typically covering the Brazilian Amazon. In this study, we analyse the relationships between hydro-climate, the surface water-energy partitioning and an index of regional forest cover change for the period 1981–2018. Additionally, we discretized three regions covering the Bolivian Amazon and the southern portions of the Peruvian and Brazilian Amazon due to their differences in the evolution of land use. In the Bolivian region, a high ratio of forest cover change, exceeding 40–50%, is related to a significant tendency to become water-limited. This change is associated with decreased rainfall, increased potential evapotranspiration and decreased actual evapotranspiration. Regardless of the region analysed, those that are characterized by a high ratio of forest cover change (>40–50%) show growing imbalance between increasing potential and decreasing actual evapotranspiration. However, in the Peruvian and Brazilian regions, hydro-climatic conditions remain energy-limited due to minor rainfall changes. The observed differences in surface water-energy partitioning behaviour evidence a complex dependence of both sub-regional (i.e., land cover changes) and large-scale (i.e., strengthening of the Walker and Hadley circulations) conditions. Our findings indicate a clear link between hydro-climatic changes and deforestation, providing a new perspective on their spatial variability on a sub-regional scale.