Hydrogels as drug carriers for delivery of antibiotics are rarely derived from natural molecules and often require complicated synthesis. Puerarin is an herbal natural product that can self-assemble to form pH-sensitive hydrogels without any structural modification. However, Puerarin hydrogels have poor thermostability and weak mechanical strength. In this work, we prepared N-(9-fluorenylmethoxycarbonyl)-l-phenylalanine (Fmoc-Phe-OH) nanofiber-reinforced Puerarin hydrogels with good mechanical and antibacterial properties for suitable drug carriers. The phase transition temperature of Puerarin hydrogels can be increased from 35 to 55 degrees C by adding only 6 mg/mL of Fmoc-Phe-OH. Rheological study indicated that the presence of Fmoc-Phe-OH not only increased the storage modulus of Puerarin hydrogels by five times, but also did not affect their pH sensitivity. SEM study and pore analysis indicated that Fmoc-Phe-OH nanofibers intertwined with the Puerarin assembly, leading to enhancement of mechanical property of hydrogel. Antibacterial test indicated that the reinforced Puerarin hydrogels have antibacterial property compared with Puerarin hydrogels. Based on these features, a controllable release of loaded model drug (berberine hydrochloride) can be realized from the reinforced Puerarin hydrogels, showing a good synergistic antimicrobial effect on the S. aureus.