Biochar amendment strategy has remained a good alternative for improving soil fertility and alleviating climate change challenges. There exists inadequate information on the role of biochar in influencing soil fungal community structure, especially up to a 40-year chrono-sequence cropping of plants such as cotton. This study was therefore purposed to determining the soil fungal constituents and their respective diversity in biochar-treated (0 t·ha^-1, 20 t·ha^-1 and 40 t·ha^-1) soils of different continuously cotton cropping years (5, 10, 20 and 40 years). The study applied high-throughput sequencing of the the 18S rRNA gene and integrated biostatistics-based techniques to screen the soil fungal community assemblage, key fungal species, soil fungal association structure and metabolic functions, the soil fungal constituents, their proportions and diversity in the biochar-treated cotton pots over the different continuous cropping years. The most abundant fungal phyla were Ascomycota and Basidiomycota. In addition, Aspergillus, a pathogenic fungus, was also detected in all our sequence data. Soil pH changes were also identified as a key factor in shaping soil fungal communities. Over the continuous cropping duration, the soil pH gradually declined. At 20 years of continuous cropping, the number of fungal OTU’s started to increase and their highest overall quantity was achieved at 20 years of continuous cropping, when pH was 7.3±0.1. Overall, our study findings show that biochar application over a long continuous cropping system alters soil fungal diversity and is therefore an important factor in soil quality management.