Novel antibacterial nanofiber/cotton hybrid yarns was fabricated by an efficient combination of a stepped pyramid stage free surface electrospinning and traditional cotton spinning process. In this study, nanofibers loading as well as the morphology variation of the nanofibers resulted from polymer solution concentration and applied voltage changes were investigated. The results demonstrated that the higher solution concentration led to larger nanofiber diameter, and higher applied voltage increased the loading of nanofibers in the hybrid yarns. Moreover, it was found that resultant nanofiber/cotton hybrid yarns which was contained a small amount of Triclosan, possessed excellent antibacterial activity against both Grampositive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. This novel method is promising for the large scale production of antibacterial nanofiber/cotton hybrid yarns for biomedical and antibacterial textiles applications.