There is an unmet demand for local vancomycin (VAN) delivery scaffolds with site retention and tunable release properties for cutaneous and surgical wounds. Nanofibers as drug delivery/cell regeneration promoting scaffolds offer great promise in this respect. High loading of the polar VAN into polymeric structures with tunable release properties can be achieved by simultaneous chemical bonding and polymer blending. Electrospinnable vanco-mycin-functionalized Eudragit E100 with a relatively high drug content and antibacterial activity (E-VAN) was investigated for the development of antimicrobial nanofibers with modifiable hydrophilicity, degradability, mechanical and release properties by blending with selected Eudragit polymers. A platform of fast dissolving nanofibers of value in immediate release applications and nanofibers with a wide spectrum of biphasic release profiles with either fast or sustained drug release at low, high and physiological pH for at least 7 days was generated. A selected nanofiber formulation with a relatively small size, adequate hydrophilicity, structural stability, and biphasic release profile at pH 7.4 showed high antibacterial activity against Staphylococcus aureus and healing efficacy of Staphylococcus aureus-inoculated full thickness excision wounds in a rat model. The E-VAN-based Eudragit nanofibers developed offer potential as versatile antimicrobial delivery/cell regeneration scaffolds for local applications under diverse pH conditions.