In this study, the antibacterial effect and mode of Coptidis rhizoma on Escherichia coli was evaluated by microcalorimetry coupled with chemometric techniques. Using an isothermal microcalorimeter, the metabolic profiles of E. coligrowth at 37 °C affected by 15 batches of C. rhizoma were measured. Through principal component analysis (PCA) on nine quantitative thermo-kinetic parameters obtained from the metabolic power–time profiles of E. coli, the antibacterial effects of C. rhizoma from various sources could be easily evaluated by analyzing the change of the two main thermo-kinetic parameters, growth rate constant k₂ and maximum heat-output power P²_m, in the second exponential phase of E. coligrowth. Then, hierarchical clustering analysis (HCA) was carried out on the two parameters to distinguish those C. rhizoma samples in respect to their antibacterial effects. Clear results were obtained to show that all 15 C. rhizoma samples with different antibacterial effects could be successfully grouped in accordance with their origins. Ranked in decreasing order, the antibacterial mode of C. rhizoma samples that were from Sichuan province had the strongest antibacterial effects, followed by samples from Chongqing city and Hubei province. Our results revealed that the developed microcalorimetry with chemometric techniques had the potential perspective for evaluating the effect and mode of Coptidis rhizoma and other Chinese materia medicas.