Diazotrophs make important contributions to nitrogen (N) inputs in agricultural ecosystems. However, strong evidence on the effects of conservation tillage (CT) on the coexistence and assembly of diazotrophic communities and related mechanisms is lacking. Here, we studied the impacts of long-term CT on the coexistence and assembly patterns of diazotrophic communities. Compared to traditional tillage (CK), CT significantly reduced both the N fixation rate (0–10 cm) and the alpha diversity of the diazotrophic community while increasing the density of the diazotrophic and overall bacterial communities. CT also reduced the competitive relationships within the diazotrophic community and enhanced network stability. Furthermore, diazotroph assembly was dominated by deterministic processes (68.63%) under CK, and stochastic processes (58.82%) under CT. Soil depth and total N (TN) were identified as crucial predictors shaping the assembly processes of diazotrophic communities under different tillage practices. The relative influence of stochastic processes on the diazotrophic community under CT varied more significantly with increasing soil depth. Overall, tillage practices and soil depth had significant influences on the coexistence and assembly processes of the soil diazotrophic community. Moreover, long-term CT may impact the selection of N fixation agents and the specific taxa associated with N fixers. Our results indicated that in CT systems, relatively sufficient nutrient availability leads to a reduction in interspecies competition, an increase in network stability, and a greater influence of stochastic processes on community assembly. These findings may help us better understand biological N fixation in sustainable agricultural systems.