<p>Chitosan–Schiff base derivatives have emerged as promising antimicrobial agents due to their tunable physicochemical and surface properties. To yield Schiff base derivatives, chitosan can be protonated and, subsequently, functionalized with molecules containing an aldehyde group. In the present study, we synthesized azo<i>-o-</i>vanillin Schiff base derivatives of low-molecular-weight chitosan, with varying the amount of azo<i>-o-</i>vanillin loading. The successful formation of Schiff base linkages was confirmed with the help of FTIR and CP-MAS (solid state) ¹³C NMR spectra. The thermal and morphological features of the materials were investigated using thermogravimetric analysis, derivative thermogravimetry, differential scanning calorimetry, scanning electron microscopy, and X-ray diffraction analyses. SEM revealed rough, uneven, and helical surface morphologies. The azo<i>-o-</i>vanillin functionalized chitosan derivatives also exhibited enhanced photoresponsive behavior. Increasing azo<i>-o-</i>vanillin content into the chitosan leads to the formation of imine linkages resulting in improved thermal stability and antimicrobial properties of the chitosan-azo<i>-o-</i>vanillin Schiff bases. The antimicrobial activity of the chitosan Schiff bases showed stronger inhibition of human pathogenic fungi and bacteria than clinically used antifungal and antibacterial drugs. The antibacterial and antifungal activities of chitosan-azo–Schiff base compounds were found to be associated with their ability to interfere with the cell membrane and thereby produce reactive oxygen species, leading to cell death. All the derivatives showed negligible cytotoxicity and hemolytic activity, confirming their biocompatibility. These findings highlight the potential of low-molecular-weight chitosan-azo<i>-o-</i>vanillin Schiff base materials in biomedical applications.</p> Graphical abstract <p></p>