The escalating threats of antimicrobial resistance and monkeypox virus infections to public health necessitate innovative therapeutic approaches. Developing materials with balanced photodynamic and photothermal effects for broad-spectrum drug-resistant bacteria elimination and monkeypox virus inactivation remains challenging. Herein, we prepared a series of Nile Red derivatives by a donor rotation and charge enhancement strategy, identifying 5-(dicyanomethylene)-9-[4-(bis(4-methoxyphenyl)amino)phenyl]-7a,12a-dihydro-5H-benzo[a]phenoxazine (TPAOMCN)-featuring alkoxy-triphenylamine and malononitrile, as the optimal candidate. TPAOMCN demonstrated extended near-infrared absorption, enhanced intersystem crossing (ISC) efficiency, and intense molecular motions, enabling dual-modal phototherapy. Electrospun TPAOMCN nanofibers (NFs) with submicron-scale diameter achieved >50°C temperature elevation and 30-fold reactive oxygen species (ROS) generation under irradiation, yielding >99.9% eradication of multidrug-resistant pathogens and virus. In methicillin-resistant S. aureus (MRSA)-induced wound infection and Vaccinia virus-mediated tail-scarred models, TPAOMCN NFs effectively eliminated MRSA colonies and reduced viral load through physical disruption of pathogen membranes, thermal denaturation of viral capsids, and ROS-mediated biomolecule oxidation, while suppressing inflammation and accelerating angiogenesis-mediated tissue repair. This study not only established a molecular engineering strategy for Nile Red to achieve prime PDT-PTT performance but also provided a paradigm for advancing dual-functional phototherapeutic platforms against emerging antimicrobial threats and monkeypox virus infections.