Our understanding of seasonal dynamics of fungal communities under different agricultural management practices remains limited. In this study, phospholipid fatty acid (PLFA) analysis and Illumina MiSeq high-throughput sequencing targeting fungal ITS1 region were combined to assess the responses of soil fungal communities to different tillage practices and cropping systems during the crop growing season. Bulk soils were collected from two long-term tillage practices (conventional tillage, CT and no-tillage, NT) under two cropping systems (maize (Zea mays L.)-soybean (Glycine max Merr.) rotation and continuous maize) in April, June and August of 2015 at 0-5 cm and 5-15 cm depths. Our results clearly highlighted the importance of cropping system and tillage for soil fungi; seasonal effect, by contrast, was weaker. The fungal PLFA content, used as an indicator of fungal biomass, appeared higher under continuous maize system than maize-soybean rotation system at depths of 0-5 cm and 5-15 cm, and higher in NT than CT at 0-5 cm depth, irrespective of sampling time. DNA-based sequencing results showed that soil fungal diversity (i.e. Shannon) and evenness (i.e. Pielou) indices were higher under rotation cropping system than continuous maize system at both soil depths. Soil fungal richness (i.e. Chao1) index was higher in NT than in CT at 0-5 cm depth. None of these three a diversity indices were significantly affected by sampling time. Cropping system and tillage played the most important roles in shaping soil fungal community at both soil depths. At 0-5 cm depth, there was an obvious consistent trend of enrichment from Ascomycota in maize-soybean rotation system to Basidiomycota in continuous maize system over the growing season. At 5-15 cm depth, there was an obvious consistent trend of enrichment from Tausonia and Chaetomidium in CT to Leptodontidium, Ceratobasidium in NT over the crop growing season. Notably NT favored symbiotrophs throughout the growing season, irrespective of cropping system and soil depth.