There is an urgent need to find new antibiotics to fight against the increasing drug resistance of microorganisms. A novel natural compound, Penicilazaphilone C (PAC), was isolated from a marine-derived fungus. It has displayed broad bactericidal activities against Gram-negative and Gram-positive bacteria. However, its bactericidal mechanism is still unknown. Herein, time-kill assays verified that PAC is a fast and efficient bactericidal agent. Furthermore, data from 4D label-free quantitative proteome assays revealed that PAC significantly influences over 898 proteins in Escherichia coli. Combining the results of biofilm formation, β-galactosidase measurement, TEM observation, soft agar plate swimming, reactive oxygen species measurement, qRT-PCR, and west-blotting, the mode of PAC action against E. coli was to block respiration, inhibit assimilatory nitrate reduction and dissimilar sulfur reduction, facilitate assimilatory sulfate reduction, suppress cysteine and methionine biosynthesis, down-regulate antioxidant protein expression and induced intracellular ROS accumulation, weaken bacterial chemotaxis, destroy flagellar assembly, etc., and finally cause the bacteria's death. Our findings suggest that PAC could have a multi-target regulatory effect on E. coli and could be used as a new antibiotic in medicine.