Understanding how large lakes interact with multilayer aquifers is crucial for managing water resources, especially where groundwater and surface water are closely connected. These interactions, found in many regions worldwide, are shaped by complex geological formations and dynamic groundwater flows. Investigating them requires a multidisciplinary approach that integrates (i) detailed 3D geological modeling, (ii) hydraulic characterization of the subsurface, and (iii) natural tracers to track water (and groundwater) movements and refine hydrogeological processes. This study examines the connection between Garda Lake and the surrounding Prealps and moraine-alluvial aquifers, a essential water reservoir for northern Italy. Spanning the provinces of Brescia, Mantova, and Verona, this area plays a key role in recharging the Po Basin aquifer system, making groundwater monitoring and protection necessary. To explore these dynamics, a hydrogeological conceptual model has been developed using geological surveys, official maps, and water well stratigraphy, supported by isotopic and microbiological tracer analysis. The results confirm a strong hydraulic link between the lake and aquifers. The moraine aquifer, composed of highly permeable sediments, is directly connected to the lake bottom and banks, allowing continuous water exchange. Below it, the alluvial multilayer aquifer, with alternating coarse and fine sediments, also interacts with the lake. These aquifers, in turn, connect to the fractured Prealps formations, forming a continuous hydrogeological system at the regional scale. By integrating geological, hydraulic, and tracer-based analyses, this study provides new insights into lake-aquifer interactions in complex settings. Beyond its relevance for the Po Plain, the approach offers a valuable framework for managing groundwater in similar regions worldwide, where lakes and aquifer plays fundamental function in the storage of this vital resource.