Soil microbiology has entered into the big data era, but the challenges in bridging laboratory‐, field‐, and model‐based studies of ecosystem functions still remain. Indeed, the limitation of factors in laboratory experiments disregards interactions of a broad range of in situ environmental drivers leading to frequent contradictions between laboratory‐ and field‐based studies, which may consequently mislead model development and projections. Upscaling soil microbiology research from laboratory to ecosystems represents one of the grand challenges facing environmental scientists, but with great potential to inform policymakers toward climate‐smart and resource‐efficient ecosystems. The upscaling is not only a scale problem, but also requires disentangling functional relationships and processes on each level. We point to three potential reasons for the gaps between laboratory‐ and field‐based studies (i.e., spatiotemporal dynamics, sampling disturbances, and plant–soil–microbial feedbacks), and three key issues of caution when bridging observations and model predictions (i.e., across‐scale effect, complex‐process coupling, and multi‐factor regulation). Field‐based studies only cover a limited range of environmental variation that must be supplemented by laboratory and mesocosm manipulative studies when revealing the underlying mechanisms. The knowledge gaps in upscaling soil microbiology from laboratory to ecosystems should motivate interdisciplinary collaboration across experimental, observational, theoretic, and modeling research.