Clotrimazole (CZ)-loaded microemulsion-containing nanofiber mats were developed as an alternative for oral candidiasis applications. The microemulsion was composed of oleic acid (0), Tween 80 (T80), and a co-surfactant such as benzyl alcohol (BzOH), ethyl alcohol (EtOH) or isopropyl alcohol (IPA). The nanofiber mats were obtained by electrospinning a blended solution of a CZ-loaded microemulsion and a mixed polymer solution of 2% (w/v) chitosan (CS) and 10% (w/v) polyvinyl alcohol (PVA) at a weight ratio of 30:70. The nanofiber mats were characterized using various analytical techniques. The entrapment efficiency, drug release, antifungal activity and cytotoxicity were investigated. The average diameter of the nanofiber mats was in the range of 105.91-125.56 nm. The differential scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD) results revealed the amorphous state of the CZ-loaded microemulsions incorporated into the nanofiber mats. The entrapment efficiency of CZ in the mats was approximately 72.58-98.10%, depended on the microemulsion formulation. The release experiment demonstrated different CZ release characteristics from nanofiber mats prepared using different CZ-loaded microemulsions. The extent of drug release from the fiber mats at 4 h was approximately 64.81-74.15%. The release kinetics appeared to follow Higuchi's model. In comparison with CZ lozenges (10 mg), the nanofiber mats exhibited more rapid killing activity. Moreover, the nanofiber mats demonstrated desirable mucoadhesive properties and were safe for 2 h. Therefore, the nanofiber mats have the potential to be promising candidates for oral candidiasis applications. (C) 2014 Elsevier B.V. All rights reserved.