Transdermal drug delivery is an attractive option for multiple disease therapies as it reduces adverse reactions and improves patient compliance. Water-dispersible β-sheet rich silk nanofiber carriers have hydrophobic properties that benefit transdermal delivery but still show inferior transdermal capacities. Thus, hydrophobic silk nanofibers were fabricated to fine-tune their size and endow them with desirable transdermal delivery capacities. Silk nanocarrier length was shortened from 2000 nm to approximately 40 nm after ultrasonic treatment. In vitro human skin and in vivo animal studies revealed different transdermal behaviors for silk nanocarriers at different nanosizes. Silk nanocarriers passed slowly through the corneum without destroying the corneum structure. Improved transdermal capacity was achieved for smaller size carriers. Both hydrophilic and hydrophobic drugs could be loaded onto silk nanocarriers, suggesting a promising future for different disease therapies. No cytotoxicity and skin irritation were identified for silk nanocarriers, which strengthened their superiority as transdermal carriers. Therefore, silk nanocarriers <100 nm may promote the percutaneous absorption of active cargos for disease therapy and cosmetic applications.