In view of the growing concern over the threat of antibiotic resistance and bacterial infections, this study evaluated the antimicrobial performance and characteristics of chitosan/polyvinyl alcohol (PVA) nanofibers incorporated with Methylene Blue (MB). Following the fabrication of chitosan/PVA nanofibers loaded with different MB concentrations via electrospinning, the samples were characterised through Field-emission Scanning Electron Microscopy (FESEM), Fourier Transform Infrared (FTIR) spectroscopy, and leaching tests. Finally, the antimicrobial inhibition level of the samples was assessed via the disc diffusion method. Based on the results, the MB-integrated chitosan/PVA nanofibers exhibited a nanoscale morphology, and the FTIR confirmed the presence of MB. The findings also established a positive correlation between the MB concentration and leaching intensity. Furthermore, the optimal antimicrobial efficacy against Escherichia coli was achieved by the chitosan/PVA/MB (5 wt.%) sample with a 2-min laser exposure, which recorded a significant inhibition zone of 8.65 mm. In conclusion, MB demonstrated potent antimicrobial properties against E. coli, suggesting its potential integration in electrospun nanofibers for combating bacterial infections via photodynamic therapy.