Assembly processes of prokaryotic and microeukaryotic community is an important issue in microbial ecology. However, unclear remains about the relative contribution of deterministic and stochastic processes to the shaping of prokaryotic and microeukaryotic communities in saline lake waters. Here, we systematically investigated the assembly processes governing the prokaryotic and microeukaryotic communities in Qinghai Lake with the use of Illumina sequencing and a null model. The results showed that both deterministic and stochastic processes play vital roles in shaping the assemblies of prokaryotic and microeukaryotic communities, in which stochastic processes appeared to dominate (> 70%). Prokaryotic communities were mainly governed by non-dominant processes (60.4%), followed by homogeneous selection (15.8%), variable selection (13.6%) and dispersal limitation (10.2%), whereas microeukaryotes were strongly driven by non-dominant processes (68.9%), followed by variable selection (23.6%) and homogenizing dispersal (6.3%). In terms of variable selection, nutrients (e.g. ammonium, dissolved inorganic carbon, dissolved organic carbon and total nitrogen) were the major factors influencing prokaryotic and microeukaryotic community structures. In summary, prokaryotes and microeukaryotes can be predominantly structured by different assembly mechanisms, in which stochasticity is stronger than deterministic processes. This finding helps to better comprehend the assembly of prokaryotic and eukaryotic communities in saline lakes.