Chitosan fiber mats were successfully processed by electrospinning. The as-spun fiber mats were neutralized with ethanol and cross-linked with glutaraldehyde. A decrease of the fiber average diameter from 243 ± 43 nm down to 215.53 nm was observed for the neutralized and cross-linking chitosan membrane. It was found that the processing conditions do not alter the initial deacetylation degree of the polymer. Polymer crystallinity index showed a decrease from 61% for the Protasan material down to 17% for the cross-linking fiber mats. A swelling index up to 1000% was observed for the cross-linked samples. Preliminary MC-3T3-E1cell culture results showed good cell adhesion and proliferation in the cross-linked chitosan fiber mats.