This work investigated the spectrum-effect relationships between HPLC fingerprints and the anti-bacterial activities of EtOAc extracts from Radix Isatidis. Fingerprints of EtOAc extracts of Radix Isatidis from various sources were established by a High-Performance Liquid Chromatography. The process of Escherichia coli (E. coli) growth affected by EtOAc extracts was monitored using a Thermal Activity Monitor (TAM) Air Isothermal Calorimeter by microcalorimetry. By analyzing the power-time curves, quantitative parameters, such as growth rate constant k, maximum heat-production rate P(m), appearance time t and total heat-production Q were obtained to characterize the interactions of E. coli and the EtOAc extracts from Radix Isatidis. The HPLC fingerprints were investigated using hierarchical clustering analysis. The main thermo-kinetic parameters from the power-time curves were analyzed using principal component analysis. The spectrum-effect relationships between the HPLC fingerprints and anti-bacterial activities were analyzed with multivariant correlation analysis. Close correlation existed between the spectrum-effect relationships of the EtOAc extracts. Salicylic acid in the HPLC fingerprints might be one of the anti-bacterial components. This work provides a general model of the combination of HPLC and microcalorimetry to study the spectrum-effect relationships of EtOAc extracts from Radix Isatidis, which can be used to search for principal components of Radix Isatidis on bioactivity.