Karst aquifers are both a valuable resource for humankind and a habitat for unique biota. The quality of freshwater sources may be easily affected by natural (e.g., geology, climate, and vegetation) and anthropogenic (e.g., agriculture, livestock, and tourism) changes, particularly in karst landscapes with highly vulnerable groundwater reservoirs. We seasonally monitored nine representative freshwater sources (i.e., six springs, a well, a surface stream, and a cave stream resurgence) in the karst system of the Runcuri Plateau (KSRP) (Western Romanian Carpathians) during seven sampling campaigns in 2019–2021. We assessed how these natural and anthropogenic factors influenced the water quality based on the European and national standards for drinking water. The geological structure (i.e., tectonics and lithology) of the KSRP was reassessed, and the environmental variables of the freshwater sites were investigated in order to evaluate their impact on the physicochemical profile, the microbial contamination, and on the meiofauna presence. Multivariate statistics were performed to gain insights into the interplay among all these factors and to evaluate the self-purification capacity of the KSRP for chemical and microbial pollutants. The most relevant drivers shaping the microbial content of the freshwater sources were the altitude of the sampling sites, the normalized difference vegetation index (NDVI), and air temperature, followed by the physicochemical profile of the waters (i.e., calcium hardness, magnesium hardness, nitrites, nitrates, conductivity, phosphates, total dissolved solids, and iron concentrations). The meiofauna presence was influenced mostly by precipitation, air temperature, and NDVI. Our results reflected the effect of the geological structure and environment on water chemistry and biota assemblages. A pollutant attenuation trend was observed in discharging waters, even though the self-purification capacity of the studied karst system was not statistically supported. More investigations are needed to comprehend the processes developed in the black box of the KSRP.