The way chemicals affect soil biological properties including the enzymatic activities may reveal soil potential to maintain biochemical processes and soil fertility. The current research was conducted to investigate how soil type and depth, and the rate and application time of pretilachlor herbicide may affect soil biological properties including soil microbial biomass (SMB), soil microbial respiration (SMR), dehydrogenase (DA), and urease activities (UR) at four paddy fields in 2021 in the city of Babol, Mazandaran province, Iran. There is little related data, to our knowledge. The experiment was factorial on the basis of a complete randomized block design with four replicates. Soil physiochemical and biological properties at different depths were determined. The effects of the three experimental factors and the interaction of soil type x soil depth were significant on SMB, SMR, DA, and UR. Accordingly, the biological activities of the soils with higher organic matter were less affected by the herbicide. The deeper depths had significantly less soil biological activities than the shallower ones. Although 7 days after pretilachlor application soil biological activities increased, 14 days after herbicide use, SMB increased, SMR and UR decreased, and DA remained constant. In general, the higher use of herbicide increased soil biological activities (except UR), as linear functions of soil depth (R2 values of 0.87 to 0.99). SMB was a more appropriate indicator to estimate SMR of soil organic matter. Soil organic matter can reduce the toxicity of pretilachlor on soil biological activities.