[objectives] Fungi are important components of nest microbiota, which are closely related to the survival, reproduction, and environmental adaptation of birds. However, most of the research on the relationship between birds and microbiota is mainly focused on intestinal microbes, but few studies focused on the micro-ecological environment in the nest. So, we want to investigate the fungal community diversity in the nest of the Green-backed Tit (Parus monticolus). [Methods] From April to June 2020, in Aha Lake National Wetland Park in Guiyang, Guizhou Province, the Green-back Tits were attracted by hanging artificial nesting boxes. All nests were inspected once a week in the early breeding period, and once every 2 days after the emergence of nest materials. Microbial samples were collected from successful breeding nests and the nests without laying eggs. Based on internal transcribed spacer (ITS) sequencing technology, the differences in fungal community composition between successful breeding nests and the nests without laying eggs of Green-backed Tit were investigated. [Results] At the phylum level, the results showed that Ascomycota (98.81%) was the main phyla of successful breeding nests, Ascomycota (85.59%) and Basidiomycota (8.33%) were the main phyla of the nests without laying eggs (Fig. 2a). At the genus level, the dominant genera of successful breeding nests were Phialemoniopsis (83.04%), Aspergillus (4.75%), Arthroderma (4.29%) and Scopulariopsis (1.78%), and the dominant genera of the nests without laying eggs were Phialemoniopsis (36.06%), Aspergillus (14.53%), Penicilliu (6.22%), Trichothecium (5.80%), Debaryomyces (1.67%) and Papiliotrema (1.09%) (Fig. 2b). Alpha diversity analysis showed that the diversity and richness of fungi in the nests without laying eggs were significantly higher than that in successful breeding nests (P < 0.05) (Fig. 3). Beta diversity analysis showed that there were significant differences in fungal communities between successful breeding nests and the nests without laying eggs (Fig. 4). A total of 19 biomarkers with statistical differences were detected by LEfSe (Linear discriminant analysis effect size) analysis. The significant biomarkers of successful breeding nests and the nests without laying eggs were distributed in Ascomycota and Basidiomycota, and there were significant differences between these two biomarker species (Fig. 5). [Conclusion] In general, by comparing the differences in fungal composition in successful breeding nests and the nests without laying eggs, we find that birds may affect the composition structure of microbiota in the nests during breeding, showing more potential pathogens in the nests without laying eggs than in successful breeding nests.