Chloramphenicol, antibiotic drug is widely used in the developing countries, may cause bone marrow suppression during its topical treatment and affecting red blood cells. Because the accumulation of high dose of chloramphenicol is necessary for this side effect, controlling its release rate is a possible approach to reduce the risk of its topical application. In the present work, chloramphenicol was encapsulated into gelatin electrospun fibers that cross-linked with non-toxic cross linker. beta-cyclodextrin, cyclic oligosaccharides of 7 membered-sugar ring, was selectively oxidized to cleave the carbon carbon bond in positions C-2 and C-3 of the glucose units to yield two aldehyde groups and end up with poly aldehyde beta-cyclodextrin (PA-beta-CD). Matrix was designed to control chloramphenicol release rate from nontoxic and biodegradable electrospun mat for topical applications. Bead-free electrospun fibers were produced at the concentration of 24 % w/v gelatin solution at 15 kV with gap distance of 17 cm with a flow rate of 2 ml/hr. Electrospun fibers were produced in the nanoscale (450-150 nm). 6 % of chloramphenicol was chosen to carry out the cross-linked matrices by using PA-beta-CD. The encapsulation efficacy was investigated by UV-isible spectroscopy and the antimicrobial activities were demonstrated against Staphylococcus aureus (gram positive) and Pseudomonas aeruginosa (gram negative bacteria) and Candida albicans. The results showed that the samples have a strong inhibitory activity against all pathogenic microorganisms used.