Microbial processes play a crucial role in the natural attenuation and engineered remediation of groundwater pollutants, yet their spatial distribution and metabolic dynamics remain challenging to monitor non-invasively. Recent advancements have highlighted a strong correlation between spectral induced polarization (SIP) signals and the presence and metabolic activity of microbial communities. The fundamental mechanisms underlying these responses, particularly those involving nanoscale microbial–mineral interactions, remain poorly understood. A critical area of inquiry is whether SIP signals are generated directly by (individuals or groups of) microbial cells or through their interactions solid interface. This study investigates the use of SIP as a geophysical tool to detect and quantify microbial metabolic activity under controlled conditions. Laboratory-scale experiments were conducted with Shewanella oneidensis MR-1, Pseudomonas sp., and a mixed culture of iron-dependent denitrifiers (KS) in an inert porous medium and in retentostat cell-suspension systems. To isolate the effects of different bacterial strains compositions on SIP responses, we synthesized alginate beads that do not contribute to polarization as a synthetic porous medium and used a retentostat setup to ensure homogenous cell mixing in suspension. In parallel incubations, we quantify molecular-biological markers, colony-forming units (CFUs), and adenosine triphosphate (ATP) to track microbial abundance and activity. The temporal evolution of polarization spectra closely followed a classic microbial growth curve: SIP responses increased during the logarithmic growth phase, correlating more strongly with metabolic activity (ATP) than with cell abundance. These findings provide compelling evidence that microbial activity itself contributes directly to measurable SIP signals. This work advances the mechanistic understanding of bio-geoelectrical coupling and supports the development of SIP-based, non-invasive monitoring tools for targeted observation of microbially mediated reactions in groundwater remediation.