This study applied an integrated DIA proteomics and untargeted metabolomics approach to investigate the molecular responses of Microsporum canis following methylene blue-mediated photodynamic therapy (MB-PDT). The combined datasets revealed widespread and coordinated changes across protein expression and metabolite pools. MB-PDT induced a systemic, multi-targeted disturbance of fungal physiology that converges on cellular energetics, proteostasis, membrane integrity, virulence determinants, and stress-response networks. Key mechanisms included disruption of membrane integrity, downregulation of virulence factors like secreted proteases, and impairment of the antioxidant defense system. The findings suggest that MB-PDT exerts its fungicidal effect through a multi-pronged mechanism, providing novel insights and supporting its development as an effective therapy for dermatophytosis.