Soil biota is the living component of soil organic matter (SOM), and plays a key role in the decomposition of SOM. Both soil biota and SOM are indicators of soil fertility and soil quality. However, they both are sensitive to soil disturbance. Although researchers developed various technologies to detect soil biota and SOM, they are mostly destructive and cause disturbance to soil, which may not reflect the actual situation of soil biota and SOM. Therefore, here we mostly focused on the non-destructive physical methods for estimating soil biota and SOM and discussed their advantages and disadvantages. These methods include but not limited to acoustic detection, radio frequency identification, radioactive tagging, hyperspectral sensing and electron energy loss spectroscopy. In addition, we pointed out the current research problems and the potential research directions for applications of physical methods in estimation of soil biota and SOM.