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Fighting against multidrug-resistant bacteria requires reliable methods to evaluate the effect of antibacterial agents. As a universal, non-destructive, and highly sensitive tool, microcalorimetry has been used in many biological investigations to provide continuous real-time monitoring of the metabolic activity. This method, based on heat-flow output, was used to evaluate the influence of two flavonoid compounds (liquiritigenin and liquiritin) on Escherichia coli. Some crucial information, such as the thermogenic power-time curve and thermokinetic parameters of E. coli growth affected by the two compounds, was obtained and further studied by chemometric techniques including similarity analysis, multivariate analysis of variance, and principal component analysis. By comparing the values of two main parameters, k (growth rate constant of the second exponential phase) and Q (heat output of the first exponential growth phase) of E. coli based on the box and whisker plot, liquiritigenin and liquiritin could be differentiated according to their antibacterial effects; liquiritin with IC (half-inhibitory concentration) of 198.6 μg mL expressed a stronger antibacterial effect than liquiritigenin with IC of 337.8 μg mL. The glucoside group in liquiritin containing four additional free hydroxyls in the diphenylpropane skeleton was crucial for inducing the antibacterial effect. Liquiritin might be a promising candidate against E. coli. This study provides a valuable method for searching for novel antibacterial agents using microcalorimetry with chemometrics. [ABSTRACT FROM AUTHOR]