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In the present condition of COVID-19, the demand for antimicrobial products such as face masks and surgical gowns has increased. Because of this increasing demand, there is a need to conduct a study on the development of antimicrobial material. Therefore, this study was conducted on the development of Aloe Vera and Polyvinyl Alcohol (AV/PVA) electrospun nanofibers. Four different fibers were developed by varying the concentrations of Aloe vera (0.5%, 1.5%, 2.5%, and 3%) while maintaining the concentration of PVA constant. The developed samples were subjected to different characterization techniques such as SEM, FTIR, XRD, TGA, and ICP studies. After that, the antimicrobial activity of the developed Aloe Vera/PVA electrospun nanofibers was checked against Gram-positive (Staphylococcus aureus) bacteria and Gram-negative (Escherichia coli) bacteria. The developed nanofibers had high profile antibacterial activity against both bacteria, but showed excellent results against S. aureus bacteria as compared with E. coli. These nanofibers have potential applications in the development of surgical gowns, gloves, etc.