The increasing environmental pollution caused by metal nanoparticles (NPs) has become a major concern, among which the ecological toxicity of zinc oxide (ZnO) NPs attracts particular attention. In this study, the Yellow River Estuary sediment was used as a research focus. A noncontact conductivity method was used to enable real-time, nondestructive monitoring of the growth kinetics of Bacillus licheniformis and Vibrio parahaemolyticus. Combining a noncontact conductivity method with high-throughput sequencing systematically revealed the multilevel toxic effects of ZnO NPs, from individual strains to the microbial community. The results showed that ZnO NPs prolonged the growth lag phase and reduced the maximum metabolic activity of both strains. These inhibitory effects were more pronounced at higher concentrations. At 160 mg/L ZnO NPs, the cell wall integrity of B. licheniformis and V. parahaemolyticus decreased by 21.99% and 12.38%, respectively. In addition, the lactate dehydrogenase release significantly increased (p < 0.01), and the intracellular reactive oxygen species levels were 15.34 times and 4.27 times those of the control group. Microbial analysis revealed that ZnO NPs significantly reduced the α-diversity index (with Chao and Shannon indices decreasing by 55.35% and 18.11%, respectively) and altered the composition of functional bacteria. This study integrated multiple methods to elucidate multilevel mechanisms, providing critical data and theoretical support for nanomaterial ecological risk assessment in estuarine environments.
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