The antibacterial activity of vapour-phase cinnamaldehyde fumigation against Staphylococcus aureus (S. aureus) was studied by plate fumigation method and colony transfer method. The effect of cinnamaldehyde on bacterial morphology and intracellular biomacromolecules was investigated by transmission electron microscopy (TEM) and laser tweezers Raman spectroscopy (LTRS)， respectively. The results showed that cinnamaldehyde had good gas-phase antibacterial activity against S. aureus， with the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 0.25 μL/mL and 0.5 μL/mL， respectively. After fumigating with cinnamaldehyde， the surface of the cells shrank. The high concentration of cinnamaldehyde may lead to the leakage of intracellular substances. The results of LTRS showed that the content of intracellular biomacromolecules increased when cinnamaldehyde was below the MBC， but decreased when cinnamaldehyde was above the MBC. With cinnamaldehyde treatment， the main chain， hydrogen bond system and spatial structure of bacterial proteins were changed， DNA single and double strands were broken， fatty acyl chain backbones were broken or cross-linked， and the order of the body lipid bilayer also changed， which affecting the fluidity of the membrane， as a result， the bacteria was inhibited in growth or killed. The results of this research showed that LTRS could detect the changes of biomacromolecules in bacteria at the single-cell level. The skeleton and structural changes of biomacromolecules in a single cell can be analyzed to infer antibacterial substances by analyzing the Raman shift and peak intensity changes. It provides a new method for studying the antibacterial mechanism of antibacterial substances on single cells.