The strong toxicity of pathogenic bacteria has resulted in high levels of morbidity and mortality in the general population. Developing effective antibacterial agents with high efficacy and long activity is in great demand. In this study, the microcalorimetric technique based on heat output of bacterial metabolism was applied to evaluate the effect of berberine on Escherichia coli, Bacillus subtilis, individually and in a mixture of both using a multi-channel microcalorimeter. The differences in shape of the power-time fingerprints and thermokinetic parameters of microorganism growth were compared. The results revealed that low concentration (20 mu g/mL) of berberine began to inhibit the growth of E. coli and mixed microorganisms, while promoting the growth of B. subtilis; high concentration of berberine (over 100 mu g/mL) inhibited B. subtilis. The endurance of E. coli to berberine was obviously lower than B. subtilis, and E. coli could decrease the endurance of B. subtilis to berberine. The sequence of half-inhibitory concentration (IC50) of berberine was: B. subtilis (952.37 mu g/mL) > mixed microorganisms (682.47 mu g/mL) > E. coli (581.69 mu g/mL). Berberine might be a good selection of antibacterial agent used in the future. The microcalorimetric method should be strongly suggested in screening novel antibacterial agents for fighting against pathogenic bacteria.