Talaromyces marneffei (TM) is an opportunistic pathogenic fungus that mainly infects immunocompromised patients. Currently, the global prevalence of talaromycosis caused by TM is increasing, leading to an increased demand for anti-TM drugs. In our previous study, a novel 28-membered macrolide compound, antifungalmycin B (ANB), was isolated from Streptomyces hiroshimensis GXIMD 06359, exhibiting significant antifungal properties. However, its in vivo mechanisms and direct antifungal effects warrant further investigation. In this study, we employed a mouse model in conjunction with transcriptomic and proteomic approaches to explore the antifungal activity of ANB against T. marneffei. In an in vivo mouse model infected with T. marneffei infection, ANB significantly reduced fungal burdens in the liver, spleen, lungs, and kidneys. Additionally, it markedly decreased the levels of reactive oxygen species (ROS) and cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Proteomic and transcriptomic studies, complemented by parallel reaction monitoring (PRM) analysis, revealed that ANB effectively disrupted acid biosynthesis and cellular energy metabolism, thereby impairing mitochondrial functions in T. marneffei. These effects were exerted through multiple pathways. These findings highlight the potential of ANB as a versatile inhibitor of polyene macrolide-resistant fungi, offering a promising therapeutic avenue for the treatment of talaromycosis.