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We investigated nozzleless ultrasound-enhanced electrospinning (USES) as means to generate nanofibrous drug delivery systems (DDSs) for pharmaceutical and biomedical applications. Traditional electrospinning (TES) equipped with a conventional spinneret was used as a reference method. High-molecular polyethylene oxide (PEO) and chitosan were used as carrier polymers and theophylline anhydrate as a water-soluble model drug. The nanofibers were electrospun with the diluted mixture (7:3) of aqueous acetic acid (90% v/v) and formic acid solution (90% v/v) (with a total solid content of 3% w/v). The fiber diameter and morphology of the nanofibrous DDSs were modulated by varying ultrasonic parameters in the USES process (i.e., frequency, pulse repetition frequency and cycles per pulse). We found that the USES technology produced nanofibers with higher fiber diameter (402 &plusmn, 127 nm) than TES (77 &plusmn, 21 nm). An increase of a burst count in USES increased the fiber diameter (555 &plusmn, 265 nm) and the variation in fiber size. The slight-to-moderate changes in a solid state (crystallinity) were detected when compared the nanofibers generated by TES and USES. In conclusion, USES provides a promising alternative for aqueous-based fabrication of nanofibrous DDSs for pharmaceutical and biomedical applications.