Antimicrobial resistance in microorganisms associated with fermented foods is increasingly recognized, yet rapid methods to characterize antibiotic response dynamics remain limited. This study evaluates antibiotic susceptibility and physiological response patterns of kombucha-associated acetic acid bacteria and motile Escherichia coli using optical nanomotion detection (ONMD), a label-free technique that quantifies single-cell mechanical activity. Two cellulose-producing species (Komagataeibacter xylinus and K. rhaeticus), one non-cellulose-producing species (K. melaceti), and E. coli were exposed to ampicillin, ciprofloxacin, and chloramphenicol. Minimum inhibitory concentrations (MICs) were determined prior to time-resolved ONMD analysis. Susceptible strains exhibited progressive suppression of confined nanomotion consistent with MIC-defined susceptibility, whereas resistant profiles maintained sustained mechanical activity. Chloramphenicol initially induced persistent or increased nanomotion at 120 min; however, extending the observation to 180 min revealed delayed suppression in susceptible strains, demonstrating that bacteriostatic antibiotics require longer observation windows for accurate ONMD classification. In motile E. coli, ONMD revealed both intracellular nanomotion puncta and swimming trajectories, which were progressively attenuated following antibiotic exposure. These findings demonstrate that ONMD complements conventional susceptibility testing by resolving time-dependent suppression of both translational motility and intracellular nanomechanical activity at the single-cell level.