To be able to predict antibiotic resistance in bacteria from fast label-free microscopic observations would benefit a broad range of applications in the biological and biomedical fields. Here, we demonstrate the utility of label-free Raman spectroscopy in monitoring the type of resistance and the mode of action of acquired resistance in a bacterial population of Escherichia coli, in the absence of antibiotics. Our findings are reproducible. Moreover, we identified spectral regions that best predicted the modes of action and explored whether the Raman signatures could be linked to the genetic basis of acquired resistance. Spectral peak intensities significantly correlated (False Discovery Rate, p < 0.05) with the gene expression of some genes contributing to antibiotic resistance genes. These results suggest that the acquisition of antibiotic resistance leads to broad metabolic effects reflected through Raman spectral signatures and gene expression changes, hinting at a possible relation between these two layers of complementary information.