Microcalorimetry was applied to study the toxic action of two cobalt compounds such as bis(salicylideniminato-3-propyl)methylaminocobalt(II) (denoted as Co(II)) and Co(III) sepulchrate trichloride (denoted as Co(sep)3+) on (E. coli) DH5α. The power-time curves of the E. coli DH5α growth were determined, and the thermokinetics parameters such as the growth rate constant k, the maximum power output Pm and the time (tm) corresponding to the Pm were obtained. The half-inhibitory concentrations (IC50) of Co(II) and Co(sep)3+ to E. coli DH5α were 15 and 42.1 mg mL−1, respectively. The experimental results revealed that the toxicity of the Co(II) compound was larger than that of Co(sep)3+. On the other hand, the scanning electron microscopy (SEM) demonstrated that the two cobalt compounds had the same toxic mechanism on E. coli DH5α, which was attributed to the damage of cell wall of the bacteria caused by both Co(II) and Co(sep)3+. Furthermore, accumulation of intracellular cobalt of E. coli DH5α, due to the interaction of Co(II) or Co(sep)3+ and E. coli DH5α, has been found by using inductively coupled plasma (ICP) analytical technique.