The objective of this research was to determine the morphology and antimicrobial activity of chitosan-cellulose acetate-gelatin blend electrospun nanofibers incorporated with lesser galangal oil by electrospinning. The conditions of applied voltage at 23 kV, flow rate at 0.7 ml/h and collector distance of 10 cm were performed. The average diameters of nanofibers incorporated with lesser galangal oil at 0.0, 0.1, 0.75, 3.0, 5.0 and 10.0%(v/v) were 152±41, 94±34, 113±43, 210 ±67, 225±56, 214±90, and 277±144 nm respectively. When adding the concentration of lesser galangal oil from 3.0 to 10.0 %(v/v), the result showed that fiber diameter increased. In addition, larger fibers with junction and non-uniform nanofibers were obtained at the concentration of lesser galangal oil of 5.0 and 10.0 %(v/v). Antimicrobial activity was investigated by dynamic shake test at different time periods by using Pseudomonas aeruginosa. The result demonstrated that the electrospun CS-CA-Gel nanofiber mats incorporated with lesser galangal oil could release bioactive reagent that effectively retarded the growth of bacteria. Therefore, the nanofibers which were fortified with lesser galangal oil have potential to be applied in various fields such as wound healing, active food packaging, drug delivery and others.