Raman spectroscopy yields a fingerprint spectrum and is of great importance in medical and biological sciences as it is non-destructive, non-invasive, and available in the aqueous environment. In this study, Raman spectroscopy and Raman mapping were used to explore the dynamic biochemical processes in screened bacteria under ceftazidime stress. The Raman spectral difference between bacteria with and without antibiotic stress was analyzed by principal component analysis and characteristic peaks were obtained. The results showed that amino acids changed first and lipids were reduced when bacteria were exposed to ceftazidime stress. Furthermore, in Raman mapping, when bacteria were subjected to antibiotic stress, the peak at 1002 cm⁻¹ (phenylalanine) increased, while the peak at 1172 cm⁻¹ (lipids) weakened. This indicates that when bacteria were stimulated by antibiotics, the intracellular lipids decreased and the content of specific amino acids increased. The reduction of intracellular lipids may suggest a change of membrane permeability. The increase of specific amino acids suggests that bacteria resist external stimuli of antibiotics by regulating the activities of related enzymes. This study explored the processes of the action between bacteria and antibiotics by Raman spectroscopy, and provides a foundation for the further study of the dynamics of microbial biochemical processes in the future.