In　this　study,　Surface-enhanced　Raman　Spectroscopy　(SERS)　and　Fourier　Transform　Infrared　Spectroscopy　(FTIR)　were　employed　to　investigate　the　molecular　changes　in　Escherichia　coli　(E.　coli)　induced　by　exposure　to　ampicillin　(AMP),　enrofloxacin　(ENR),　ciprofloxacin　(CIP),　and　norfloxacin　(NFX)　over　time.　The　optimal　concentration　of　E.　coli　for　SERS　analysis　was　determined　to　be　50　μL　of　bacterial　suspension,　diluted　six　times　to　achieve　an　OD600　≈　0.1.　The　primary　changes　in　the　SERS　spectra　were　observed　at　1267　cm−1,　corresponding　to　the　amide　III　band　in　proteins,　while　the　FTIR　spectra　revealed　significant　changes　in　the　1200–900　cm−1　range,　associated　with　carbohydrates,　under　AMP　treatment.　ENR,　CIP,　and　NFX,　which　are　quinolone　antibiotics,　act　as　inhibitors　of　DNA　synthesis.　The　main　changes　in　the　SERS　spectra　for　antibiotic-resistant　E.　coli　were　observed　at　760　cm−1　(attributed　to　cytosine　and　uracil),　960　cm−1　(C[sbnd]N　stretching　and　C[dbnd]C　deformation),　and　1140　cm−1　(C[sbnd]O[sbnd]C　stretching　and　ring　breathing).　In　the　FTIR　spectra,　significant　changes　were　detected　at　1655　cm−1,　1544　cm−1,　and　1239　cm−1,　corresponding　to　the　amide　I,　amide　II,　and　amide　III　bands,　respectively.　The　combination　of　SERS　and　FTIR　with　principal　component　analysis　(PCA)　enabled　the　detection　of　molecular　modifications　in　antibiotic-resistant　E.　coli　exposed　to　different　classes　of　antibiotics.　These　findings　enhance　our　understanding　of　the　mechanisms　of　action　of　antibiotics　in　bacteria.　©　2025　Elsevier　B.V.