The objective of this research was to produce antimicrobial gelatin nanofibers by electrostatic spinning. The antimicrobial compound contained in the nanofibers was nisin which has the Minimum Inhibitory Concentration (MIC) of 0.05 mg/mL. After heated at 120 ℃ for 30 minutes, nisin in acid solution (pH 2) still retained its antimicrobial activity. Gelatin-nisin solutions (pH ~ 3) were prepared in acetic acid: water (70:30 v/v). Viscosity and conductivity of the solutions increased as the concentration of gelatin and nisin increased. Only nanofiber made from 22% and 24% w/v gelatin concentration was smooth without beads on string. Nisin concentration did not affect the average diameter of nanofiber, while increasing gelatin concentration caused an increase in nanofiber average diameter. The antimicrobial gelatin nanofiber mats were crosslinked by saturated glutaraldehyde vapor at 37 ℃ for 5 minutes. Tensile strength, Young’s modulus and elongation of nanofiber mats (47.8 um – 132.8 um) decreased with increasing initial nisin concentration. Tensile strength and elongation of nanofiber mates (3% w/w nisin concentration and 20% - 24% w/v gelatin concentrations) were not significantly different. While their Young’s moduli were significantly different. Increasing initial nisin concentration in the nanofiber increased the width of inhibition zone of Lactobacillus plantarum TISTR 850. Antimicrobial gelatin nanofiber which was produced from gelatin-nisin solution with different gelatin concentrations showed no significant difference in inhibition zone width. The nisin retention in crosslinked antimicrobial gelatin nanofiber mat was 1.08% - 1.22% w/w. Effects of temperature and water activity (aw ) on nisin release from crosslinked antimicrobial gelatin nanofiber mat were investigated on nanofiber that was produced from 22% w/v gelatin solution with 3% w/w nisin concentration. It was found that increasing temperature in the 5 ℃ - 45 ℃ range caused an increase in nisin release but increasing aw in the 0.955 – 0.992 range caused no significant difference in the release. Crosslinked antimicrobial gelatin nanofiber mat showed an inhibition effect on Staphylococcus aureus and Listeria monocytogenes but not Salmonella Typhimurium. After storage at 25 ℃ for 5 months, the crosslinked antimicrobial gelatin nanofiber mat still retained its inhibition activity against Lactobacillus plantarum.