Transfersomes are deformable vesicles that can transport drugs across difficult-to-permeate barriers in human tissues. In this work, nano-transfersomes were produced for the first time by a supercritical CO2assisted process. Operating at 100 bar and 40 °C, different amounts of phosphatidylcholine (2000 and 3000 mg), kinds of edge activators (Span®80 and Tween®80), and phosphatidylcholine to edge activator weight ratio (95:5, 90:10, 80:20) were tested. Formulations prepared using Span®80 and phosphatidylcholine at an 80:20 weight ratio produced stable transfersomes (−30.4 ± 2.4 mV ζ-potential) that were characterized by a mean diameter of 138 ± 55 nm. A prolonged ascorbic acid release of up to 5 h was recorded when the largest amount of phosphatidylcholine (3000 mg) was used. Moreover, a 96% ascorbic acid encapsulation efficiency and a quasi-100% DPPH radical scavenging activity of transfersomes were measured after supercritical processing.