Objective: This study used nontargeted metabolomics to characterize metabolic alterations in fungal ball sinusitis (FBS), identify differential metabolites, and explore their roles in fungal infection, immune dysregulation, and histopathological mechanisms. Methods: This study prospectively enrolled 30 patients with FBS and 29 controls with isolated nasal septum deviation who underwent surgery at Beijing Tongren Hospital between February 2023 and April 2024. Specimens of collected fungal balls and secretions of the middle meatus were analyzed for metabolites. Metabolomic profiling was performed using LC-MS/GC-MS. Metabolite identification was achieved through cross-referencing with the HMDB and METLIN databases. We use the package "ropls" in R language to perform multivariate statistical analysis, Principal Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), detecting differential metabolites (VIP >1, p < .05). Metabolic pathway enrichment analysis was performed based on the KEGG database. Results: In the positive ionization mode, seven differentially expressed metabolites were identified, such as oxidized L-glutathione. In the negative ionization mode, six differentially expressed metabolites were identified, such as 4-acetamidobutanoic acid, lysophosphatidylcholine (LPC) O-18:0, LPC O-20:1, and LPE O-20:1. Comparative analysis revealed upregulation of LPC O-18:0, LPC O-20:1, and LPE O-20:1 in fungal ball specimens, while oxidized L-glutathione and 4-acetamidobutanoic acid showed relative downregulation. At the same time, significant dysregulation was observed in the nucleotide metabolism pathways within fungal ball samples. Conclusion: The composition of metabolites and metabolic pathways within the fungal ball in the affected sinus cavity of FBS exhibits significant differences compared to secretions from the middle meatus on the affected side, the healthy side, and the control group.