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Groundwater is an important freshwater resource and hosts specialized microbial assemblages providing fundamental ecosystem services. The current knowledge on the role of aquatic microorganisms in subsurface ecosystems is still limited. This work aimed to explore the links between groundwater hydrogeochemical properties and microbial community traits in a volcanic unconfined aquifer, moderately impacted by anthropic activities. The main physical and chemical parameters of groundwater samples were analyzed, along with microbial biomass (total cell counts, ATP-active biomass concentration), potential metabolic activity, and physiological profiles at the microbial community level (Biolog ™ EcoPlates). The results showed the coexistence of oxidizing and reducing groundwater conditions across the study area. We discriminated two groups of oxidizing/reducing groundwater samples (Ox and Red), each including two subgroups with different chemical conditions attributed to contrasting levels of anthropogenic impact for non-intensive agricultural practices and waste disposal activities (Ox − and Ox + ; Red − and Red +). Although the microbial biomass was likely not affected by changing redox, the microbial metabolic potential and functional diversity changed significantly. In the Ox samples, the community-level physiological profiles were different, mainly owing to the utilization of carboxylic acids (Ox − > Ox +) and carbohydrates (Ox + > Ox −). In the Red samples, a wider set of organic substrates were consumed by the microbial communities, including those less bioavailable (e.g., phenols). Significant differences were also found between Red − and Red + , mainly owing to the relative increase in the utilization of amino acids in Red − , polymers and amines in Red + , along with the active biomass. By reflecting the local redox conditions and moderate levels of anthropogenic impact, the applied approach highlighted changes of microbial metabolic potential and physiological profiles that imply direct repercussions on biogeochemical cycling and the ecosystem services provided by groundwater microorganisms. [ABSTRACT FROM AUTHOR]