Clostridium difficile (C. difficile) infection (CDI) rates have exhibited a steady rise worldwide over the last two decades and the infection poses a global threat due to the emergence of antibiotic resistant strains. Interstrain antagonistic interactions across the host microbiome form an important strategy for controlling the emergence of CDI. The current diagnosis method for CDI, based on immunoassays for toxins produced by pathogenic C. difficile strains, is limited by false negatives due to rapid toxin degradation. Furthermore, simultaneous monitoring of nontoxigenic C. difficile strains is not possible, due to absence of these toxins, thereby limiting its application toward the control of CDI through optimizing antagonistic interstrain interactions. Herein, we demonstrate that morphological differences within the cell wall of particular C. difficile strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance. As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed C. difficile samples. The sensitivity of this contact-less electrophysiological method is benchmarked against the immunoassay and microbial growth rate methods for detecting alterations within both, toxigenic and nontoxigenic C. difficile strains after vancomycin treatment. This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes.