This thesis investigates the effects of agricultural practices on soil microbial communities, with a specific focus on understanding how different tillage practices and crop rotation impact soil fungal communities within the distinct soil fractions. A two consecutive year study (2021 and 2022) was conducted in three fields located in Manitoba, Canada, each undergoing a different phase a crop rotation cycle that included corn, canola, and soybean. Additionally, the research examined multiple tillage practices that included conventional tillage, deep tillage, vertical tillage, and raised bed practice. To comprehensively assess soil fungal microbial communities, next-generation sequencing of the internal transcribed spacer region within nuclear ribosomal DNA was employed. Our study confirmed that tillage practices and crop rotation shape the structure of the soil fungal communities within the different soil fractions. In both years, Ascomycota and Basidiomycota were recognized as the prevailing fungal phyla in all tillage practices and crop rotations. These two phyla are widely recognized as the primary classical fungal decomposers and pathotrophs in soils. Furthermore, while tillage practices did not exert a significant impact on soil fungal alpha diversity, the highest predicted average fungal richness, as measured by Observed, Shannon, and Simpson alpha diversity indices, was associated with vertical tillage in 2021 and conventional tillage in 2022. Moreover, there was a significant difference in soil fungal alpha diversity among plots within different fields in 2021. Predictions based on both Shannon and Simpson indices indicated that plots in soybean in 2021 and plots in corn in 2022 were anticipated to exhibit the highest average fungal richness. We also noted a significant difference in soil fungal alpha diversity among different soil fractions in both years of our study. The relative abundance of numerous amplicon sequence variants exhibited significant difference in response to both tillage practices and crop rotation. Specifically, soils subjected to conventional tillage practices demonstrated a significantly lower relative abundance of plant pathogens within the phylum Ascomycota and a higher relative abundance of saprotrophic fungi. Our investigation further revealed that the relative abundance of beneficial soil fungi, particularly arbuscular mycorrhizal fungi, was higher in soils under the vertical tillage practice. In plots within the corn field, we also noted a markedly higher relative abundance of arbuscular mycorrhizal fungi. Conversely, plots within canola fields exhibited a higher relative abundance of saprotrophic fungi, including members of the phyla Ascomycota, Basidiomycota, and Mortierellomycota. Our results suggest that tillage and crop rotation influence soil fungal community distribution. This study provides insights into the intricate relationships between agricultural practices and soil fungal communities, shedding light on the dynamics of these critical components of agricultural ecosystems in Manitoba.