As a widely used traditional Chinese medicine (TCM), Radix Aconiti Lateralis Preparata (Fuzi) is not only efficacious but also poisonous. Its toxicity and processed products should be taken into account and effectively evaluated. In this study, a non-invasive and non-destructive microcalorimetric method was employed to evaluate and compare the toxicity of Fuzi and its three processed products including Yanfupian, Heifupian, and Danfupian on Escherichia coli (E. coli). Some important metabolic information, such as the power-time curves and some quantitative thermokinetic parameters including growth rate constant k, heat output power P, inhibitory ratio I, and half inhibitory concentration IC50, of E. coli growth affected by various concentrations of Fuzi and its processed products were obtained. Combined with chemometric techniques including multivariate analysis of variance and principal component analysis on this information, it could be seen that Fuzi and its processed products could be distinguished according to their toxic effects on E. coli. The IC50 values of 14.6 mg/mL for Fuzi, 59.2 mg/mL for Yanfupian, 118.3 mg/mL for Heifupian, and 182.7 mg/mL for Danfupian illustrated that the sequence of toxicity on E. coli was Fuzi > Yanfupian > Heifupian > Danfupian. This study provided a useful method and idea of the combination of microcalorimetry and chemometrics for studying the toxic effects of TCMs and other substances.