Pulsed electric fields (PEF) as a new pasteurization technology played an important role in the process of inactivating microorganisms. At the same time, temperature could promote the process of electroporation, and achieve better inactivation effect. This article studied the inactivation effect of PEF on Saccharomyces cerevisiae, Escherichia coli, and Bacillus velezensis under different initial temperatures (room temperature-24°C, 30°C, 40°C, 50°C). From the inactivation results, it found temperature could reduce the critical electric field intensity for microbial inactivation. After the irreversible electroporation of microorganisms occurred, the nucleic acid content and protein content in the suspension increased with the inactivation rate because the cell membrane integrity was destroyed. We had proved that the electric field and temperature could promote molecular transport through the finite element simulation. Under the same initial temperature and electrical parameters (electric field intensity, pulse width, pulse number), the lethal effect on different microorganisms was Saccharomyces cerevisiae > Escherichia coli > Bacillus velezensis.