Objectives: This study compared the proteomics of Escherichia coli containing the multidrug resistance plasmid pEK499 under antimicrobial stress and with no antimicrobial. Methods: We utilised mass spectrometry-based proteomics to compare the proteomes of the bacteria and plasmid under antimicrobial stress and no antimicrobial. Results: Our analysis identified statistically significant differentially abundant (SSDA) proteins common to groups exposed to the β-lactam antimicrobials but not ciprofloxacin, indicating a β-lactam stress response to exposure from this class of drugs, irrespective of β-lactam resistance or susceptibility. Data arising from comparisons of the proteomes of ciprofloxacin-treated E. coli and controls detected an increase in the relative abundance of proteins associated with ribosomes, translation, the TCA cycle and several proteins associated with detoxification, and a decrease in the relative abundance of proteins associated with the stress response, including oxidative stress. We identified changes in proteins associated with persister formation in the presence of ciprofloxacin but not the β-lactams. The plasmid proteome differed across each treatment and did not follow the pattern of antimicrobial–antimicrobial resistance (AMR) protein associations: a relative increase was observed in the amount of CTX-M-15 in the presence of cefotaxime and ciprofloxacin, but not the other β-lactams, suggesting regulation of CTX-M-15 protein production. Conclusion: The proteomic data from this study provided novel insights into the proteins produced from the chromosome and plasmid under different antimicrobial stresses. These data also identified novel proteins not previously associated with AMR or antimicrobial responses in pathogens, which may well represent potential targets of AMR inhibition.