Fallow rotation has profound significance for sustainable agriculture in China. Currently, winter fallow system such as tillage and sunning, growing winter green manure crops, and intercropping with bean crops are practiced and advocated in mono-cropping obstacle area. Planting new varieties of crops, soil amelioration, scientific irrigation, and control of crop uptake of heavy metals are carried out in heavy metal pollution area. The seasonal fallow and annual fallow are enforced in groundwater funnel area. Significant economic, social and ecological benefits have been reachieved after adoption and implementation of the rotation/fallow modes in three typical regions. A field experiment was conducted to examine the effects of fallow (CK), residual plastic film mulching, deep tillage in hot summer, organic manure application, straw retention, and planting green manure on soil microbial biomass carbon and nitrogen, enzyme activity and fungal community in major ecological degradation area of Gansu Province. Soil microbial biomass and enzyme activity were analyzed while soil microbial DNA was extracted for PCR amplification and high throughput sequencing. The results showed that different treatments had significant effects on soil microbial biomass carbon and nitrogen, urease, alkaline phosphatase, dehydrogenase, organic matter and pH value except for catalase activity. The top three most abundant phyla were Ascomycota, Basidiomycota, and Mortierellomycota. The relative abundance of Ascomycota in three consecutive years deep tillage in hot summer treatment and three consecutive years fallow after corn straw retention 7500 kg/hm² and 30 cm deep tillage treatment were significantly higher (43.23% and 69.38%, respectively) than CK (33.71%). Dothideomycetes, Sordariomycetes, Agaricomycetes, and Mortierellomycetes were the dominant fungus, with the relative abundance of Dothideomycetes being the highest (60.69%) in three consecutive years fallow after corn straw retention 7500 kg/hm² and 30 cm deep tillage treatment while Sordariomycetes being the highest in other treatments (4.11%-24.79%), respectively. The diversity indexes of Simpson and Shannon-wiener in returning green manure of Pisum sativum (seed dressing with rhizobia 8.5 g/kg) for 3 consecutive years after corn straw retention 7500 kg/hm² and 30 cm deep tillage treatment, three consecutive years fallow after corn straw retention 7500 kg/hm² and deep tillage of 30 cm treatment, returning green manure of Vicia sativa for 3 consecutive years after corn straw retention 7500 kg/hm² and 30 cm deep tillage treatment were significantly lower than CK and other treatments. The abundance indexes of ACE and Chao1 in returning green manure of Iicia villosa for 3 consecutive years after corn straw retention 7500 kg/hm² and 30 cm deep tillage treatment were significantly higher than CK and other treatments. Saprotroph was the main functional group of soil fungi in three consecutive years fallow after corn straw retention 7500 kg/hm² and 30 cm deep tillage treatment (62.9%), whereas pathotroph and saprotroph were the main functional group of soil fungi in other treatments. Redundancy analysis (RDA) and the Monte Carlo permutation test showed that the soil microbial biomass carbon, soil microbial biomass nitrogen, pH value, and organic matter content showed a significant impact on the composition of the fungal community. Compared to CK, residual plastic film mulching and deep tillage in hot summer treatments, combining organic manure application, straw retention and returning green manure with deep tillage could reduce pathotroph relative abundance, which was conducive to the maintainance of the soil ecosystem health.