Microbial motility is a reliable biosignature of extant life, serving as a measurable indicator of active metabolism, structural integrity, and environmental responsiveness — a relationship recognized since Leeuwenhoek’s earliest observations and regularly used in modern life-detection strategies. Shewanella oneidensis MR-1’s unique capacity for extracellular electron transport (EET) makes it an ideal model organism for investigating how an indium tin oxide (ITO) electrode functioning as an insoluble electron acceptor (IEA) stimulus can modulate microbial motility and improve biosignature detection. Prior studies characterizing electrokinesis, energy taxis, and congregation behaviors in MR-1 have been limited to two-dimensional observations, which fault to capture the full spatial complexity of microbial swimming and often constrain organisms to surface-influenced environments inconsistent with natural conditions. We developed a custom Mach-Zehnder Digital Holographic Microscope (MZ-DHM) paired with an electrochemical sample chamber to characterize the three-dimensional motility response of S. oneidensis MR-1 to applied electric potentials under anaerobic conditions. Motility metrics — including swimming speed, number of motile cells, and reversal rates — were quantified as a function of both electrode activation (0.0 V vs. 0.6 V) and distance from the ITO electrode surface across four independent trials. Applying 0.6 V to the working electrode produced a 3.6-fold increase in the total number of motile cells (57 to 208) over four trials and a 2.6-fold increase in average swimming speed [± SD of instantaneous speeds] (13.2 ± 11.6 μm/s to 33.8 ± 18.6 μm/s). Both motile cell counts and swimming speeds showed a clear spatial gradient, with the strongest responses concentrated within 50 μm of the electrode surface and progressively declining with distance, this is consistent with previous IEA stimuli studies using S. oneidensis but now resolved in three dimensions. The total number of reversal events as well as the reversal rate per microbe increased near the electrode surface. This indicates that electrokinesis and congregation are true phenomenon and are not just a aberration due to the two-dimensional. These results validate the use of IEAs as a reliable stimulus method for enhancing detection of motility-based biosignatures, and establish a proof-of-concept platform (i.e., DHM and electrochemical sample chamber) that is well suited for deployment in extreme terrestrial environments to understand the impacts of IEAs on extreme environmental samples.