This study provides an eco-friendly dyeing processing for chitosan fiber using Oenothera odorata jacquin as a dye. The effects of chemical mordants (Al, Cu, Fe) and natural mordant (Chestnut shell) on the color change for dyed chitosan fibers were measured by K/S values, L, a*, b*, H, V, C values, color fastness, and antimicrobial activity. The results are as follows. Dyeing conditions of Oenothera odorata jacquin on chitosan fibers were optimized to 70℃, 30 minutes and 200% on weight of fabric (o.w.f.). The premordant concentration of aluminium (Al), copper (Cu) and iron (Fe) of chitosan fibers was optimized to 3% (o.w.f.) and 1% (o.w.f.), respectively. The post-mordant concentration of chemicals, such as Al, Cu and Fe, on chitosan was determined to 1% (o.w.f.). The hue of chitosan fibers by chemical mordants was measured to be reddish & yellow. The pre-mordant concentration of Chestnut shell of chitosan was optimized to 70% (o.w.f.). The post-mordant concentration of Chestnut shell on chitosan was determined to be 50% (o.w.f.). The hue of chitosan fibers by Chestnut shell mordant was measured to be reddish & yellow. The wet cleaning fastness of chitosan fibers was improved by a pre-mordant that used chemical mordants. In the case of the Chestnut shell mordant, the wet cleaning fastness was improved by a postmordant. The dry cleaning fastness of chitosan fibers was excellent regardless of mordants and mordant methods. The antimicrobial activity of the chitosan fiber was shown at 99.9% and its excellent qualities remained after the dyeing and mordant processing.