Groundwater, a critical source of drinking water, plays an essential role in global biogeochemical cycles, yet its microbial ecosystems remain insufficiently characterized, particularly in pristine karst aquifers. This study conducted high-resolution profiling of microbial communities and environmental parameters in two representative alpine karst aquifers in Slovenia: Idrijska Bela and Krajcarca. Monthly groundwater samples from the Krajcarca spring and Idrijska Bela borehole over a 14-month period were analyzed using whole-metagenome sequencing (WMS), UV-Vis spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), and isotopic analysis. The results revealed stable hydrochemical conditions with clear spatial differences driven by bedrock composition and groundwater residence time. Bacterial communities displayed strong correlations with hydrochemical parameters, while archaeal communities exhibited temporal stability. Functional gene profiles mirrored bacterial patterns, emphasizing the influence of environmental gradients on metabolic potential. No significant temporal changes were detected across two sampling campaigns (2016–2023), highlighting the resilience of these aquifers. This work establishes a valuable baseline for understanding pristine groundwater microbiomes and informs future monitoring and water quality management strategies.