Electrospun nanofibers made from chitosan are promising materials for surgical wound dressings due to their non-toxicity and biocompatibility. However, the antibacterial activity of chitosan is limited by its poor water solubility under physiological conditions. This study addresses this issue by producing electrospun nanofibers mainly from natural compounds, including chitosan and quaternized chitosan, which enhance both its solubility for electrospinning and the antibacterial activity of the resulting electrospun nanofibers. Additionally, antimicrobial agents like caffeic acid or berberine chloride were incorporated. The glutaraldehyde-treated nanofibers showed improved mechanical properties, with an average tensile strength exceeding 2.7 MPa, comparable to other chitosan-based wound dressings. They also demonstrated enhanced water stability, retaining over 50% of their original weight after one week in phosphate-buffered saline (PBS) at 37 °C. The morphology and performance of these nanofibers were thoroughly examined and discussed. Furthermore, these membranes displayed rapid drug release, indicating potential for inhibiting bacterial growth. Antibacterial assays revealed that S2-CX nanofibers containing caffeic acid were most effective against E. coli and S. aureus, reducing their survival rates to nearly 0%. Similarly, berberine chloride-containing S4-BX nanofibers reduced the survival rates of E. coli and S. aureus to 19.82% and 0%, respectively. These findings suggest that electrospun membranes incorporating chitosan and caffeic acid hold significant potential for use in antibacterial wound dressings and drug delivery applications.