Aim To compare the effects of steam explosion and Lactobacillus buchneri inoculation on fungal community in ensiled total mixed ration (TMR) during aerobic exposure. Methods and Results The TMRs were prepared using wheat straw with or without steam explosion, sweet potato residue, lucerne hay, maize meal and soybean meal, and ensiled with or without L. buchneri inoculation. Fungal communities were detected by high-throughput sequencing. All ensiled TMRs were well ensiled and steam explosion has a major effect on improving aerobic stability. The fungal species, such as Xeromyces bisporus and Cryptococcus victoriae, that dominated in the TMR decreased after ensiling, with a concomitant increase in Candida humilis, Pichia kudriavzevii, Aspergillus flavus and Phanerochaete chrysosporium. Most mould species decreased, with C. humilis and P. kudriavzevii dominating during aerobic exposure. Conclusion Steam explosion could improve the aerobic stability in ensiled TMR by inhibition of C. humilis. Significance and Impact of the Study High-throughput sequencing used in this study provides insight into the fungal community in ensiled TMR during aerobic exposure, which could contribute towards elucidating the mechanism by which aerobic deterioration develops.