Conventional irrigation methods decrease greenhouse soil aeration, which leads to restricted root growth and reduced soil fungal abundance in greenhouse grapes. In this study, aerated irrigation equipment was used to investigate the effects of aerated irrigation on the biomass accumulation, root growth, and soil fungal community structure of grape seedlings. The results show that aerated irrigation significantly increased the root length, root surface area, root volume, and number of root tips by 38.5%, 32.1%, 62.1%, and 23.4%, respectively, at a soil depth of 20–40 cm (p ≤ 0.05). The chao1 index and ACE index of fungi at different soil depths under aerated irrigation were higher than those without aerated treatment; aerated irrigation changed the relative abundance of dominant fungi in rhizosphere soil. At a soil depth of 20–40 cm, aerated irrigation increased the abundance of Fusarium by 42.2%. Aerated irrigation also contributed to the abundance of the beneficial fungal genera Mortierella, Cladosporium, and Glomus. At a soil depth of 0–20 cm, the abundance of Mortierella in the soil that received aerated treatment was 180.6% higher than in the control treatment. These findings suggest that aerated irrigation is a promising strategy for the promotion of grape root growth and biomass accumulation, and it can also increase the abundance of some beneficial fungi.