Antibacterial agents play a crucial role in healthcare and public health by combating bacterial infections.However, the emergence of antibiotic resistance necessitates a deeper understanding of 6 their mechanisms of action.This review explores the use of spectroscopic techniques, including UV-Vis, FTIR, NMR, Raman spectroscopy, mass spectrometry, and fluorescence spectroscopy, to elucidate the mechanisms of action (MOA) of antibacterial agents in the period from 2003 to 2024.The review discusses how these techniques can elucidate the MOA of classical antibiotics, antiseptics and antimicrobial peptides.Since these spectroscopic techniques provide valuable insights into the mol. interactions between antibacterial agents and bacteria, offering a comprehensive understanding of their mechanisms at a mol. level.By studying electronic transitions, chem. bond vibrations, metabolomics, and protein interactions, researchers can gain in-depth knowledge of antibacterial MOAs.This knowledge can inform the development of improved treatments and strategies to combat bacterial resistance, leading to better patient outcomes and global public health.