Bacterial infection is associated with gastric carcinogenesis. However, the relationship between nonbacterial components and gastric cancer (GC) has not been fully explored. We aimed to characterize the fungal microbiome in GC. We performed ITS rDNA gene analysis in cancer lesions and adjacent noncancerous tissues of 45 GC cases from Shenyang, China. Obtaining the OTUs and combining effective grouping, we carried out species identifications, alpha and beta diversity analyses, and FUNGuild functional annotation. Moreover, differences were compared and tested between groups to better investigate the composition and ecology of fungi associated with GC and find fungal indicators. We observed significant gastric fungal imbalance in GC. Principal component analysis revealed separate clusters for the GC and control groups, and Venn diagram analysis indicated that the GC group showed a lower OTU abundance than the control. At the genus level, the abundances of 15 fungal biomarkers distinguished the GC group from the control, of which ( = 0.000246) and ( = 0.00341) were enriched in GC, while ( = 0.002324) and ( = 0.009158) were decreased. Combining the results of Welch's t test and Wilcoxon rank sum test, () was significantly elevated in GC. The species richness Krona pie chart further revealed that occupied 22% and classified GC from the control with an area under the receiver operating curve (AUC) of 0.743. Random forest analysis also confirmed that could serve as a biomarker with a certain degree of accuracy. Moreover, compared with that of the control, the alpha diversity index was significantly reduced in the GC group. The Jaccard distance index and the Bray abundance index of the PCoA clarified separate clusters between the GC and control groups at the species level ( = 0.00051). Adonis (PERMANOVA) analysis and ANOVA showed that there were significant differences in fungal structure among groups ( = 0.001). Finally, FUNGuild functional classification predicted that saprotrophs were the most abundant taxa in the GC group. This study revealed GC-associated mycobiome imbalance characterized by an altered fungal composition and ecology and demonstrated that can be a fungal biomarker for GC. With the significant increase of in GC, the abundance of were increased, while , and were obviously decreased. In addition, may mediate GC by reducing the diversity and richness of fungi in the stomach, contributing to the pathogenesis of GC.