In the present study, we evaluated whether long-term no-tillage with different straw mulching frequencies (zero, once, twice and three times every three years) would affect soil microbial community composition, co-occurrence patterns, and the abundances of two pathogenic fungi (Fusarium graminearum and Fusarium moniliforme). Soil samples were collected in April, June, August and October of 2017 from a long-term tillage experimental field (initiated in 2007) in northeast China. Soil microbial community composition and pathogenic fungi abundances were determined using high-throughput sequencing and quantitative PCR, respectively. The results indicated that no-tillage and straw mulching caused significant changes in bacterial and fungal relative abundances at phylum and genus level compared with conventional tillage, and more significantly changed bacterial and fungal genera were found in the mulching treatments of twice and three times every three years, respectively. Permutational multivariate analysis of variance (PERMANOVA) showed that sampling time had a very significant effect on both bacterial and fungal communities (P < 0.01), and the effects of tillage treatment on fungal and bacterial communities were very significant (P < 0.01) and significant (P < 0.05), respectively. Moreover, no-tillage and straw mulching significantly increased fungal richness compared with conventional tillage, but the effect on bacterial diversity was not significant. The results of diversity and PERMANOVA suggested that fungi are more susceptible to tillage practices than bacteria. Furthermore, molecular ecological network analysis revealed that straw mulching reduced the complexity of both bacterial and fungal networks, but increased their ratios between positive and negative links. In addition, no-tillage and straw mulching increased the abundances of F. graminearum and F. moniliforme, especially in the mulching treatments of zero and three times every three years, which might increase the risk of maize root rot. Some soil properties and microbial genera were found to be related to the abundance changes of these two pathogenic fungi. Our research will provide some guidance for no-tillage and residue management of maize cropping in northeast China.