Electrospun nanofibers (NFs) as drug delivery/tissue regeneration template and antimicrobial photodynamic therapy (APDT) have been widely investigated as two different approaches to enhance wound healing. In the present study, the two approaches were combined in a single platform for greater healing enhancement potentials. Composite photosensitizer-eluting NFs were developed using a polyhydrohybutyrate/polyethylene glycol (60:40 PHB/PEG) polymer blend and methylene blue (MB) as antimicrobial photosensitizer (PS). NFs protected the photoactivity of entrapped MB, enhanced its photodynamic activity against two wound bacteria, Staphylococcus aureus standard strain (SAst) and MRSA and sustained MB release allowing for flexible PS dosing and irradiation schedules. This combined PS-eluting NFs/APDT approach proved effective in the treatment of SAst-inoculated excision wounds in a challenging immunocompromized rat model. This was verified by morphological, morphometric, microbiological, histopathological and RT-PCR studies. Inclusion of PS-eluting NFs as an additional active component of APDT generates a combined non-antibiotic antimicrobial/cell regeneration approach with great potentials for wound healing and other biomedical applications.