The performance of transfersomes as nanovesicular systems to enhance the skin permeation of bioactive compounds may be modulated according to their composition of edge activators (EAs). Vesicle size, deformability, and encapsulation efficiency can be altered when using nonionic surfactants as EAs with opposing hydrophilic-lipophilic balance (HLB). Thus, this work aimed to assess the impact on the physicochemical properties and the in vitro cytotoxicity profile of transfersomal formulations composed of single or mixed EAs, with opposing HLB values. Transfersomes made of single or mixed nonionic surfactants (Tween® 80 and/or Span® 80) were prepared in the presence and absence of caffeine, used as a model hydrophilic compound. Single or mixed EAs-containing transfersomes displayed promising physicochemical properties for cutaneous applications and were found to be stable for one month of refrigerated storage. Moreover, all transfersomal formulations were compatible with a keratinocyte cell line upon 24 h treatment. Altogether, these preliminary data suggest that the use of mixed nonionic surfactants as EAs may be further explored to modulate the performance of transfersomes as skin permeation enhancers. Keywords: Vesicular systems, nanotechnology, nonionic surfactants, caffeine