Microplastics (MPs), recognized as emerging pollutants, pose a significant threat to diverse organisms and have adverse effects on agricultural soil. High‐density polyethylene (HDPE) holds a prominent position among prevalent forms of MPs. In the current investigations, the impact of HDPE was assessed at four different concentrations (0.25%, 0.5%, 0.75%, and 1.0%) on agricultural soil, microbial population, exoenzymes activities including amylase, cellulase, and invertase, and alteration in carbon‐to‐nitrogen (C/N) ratio. Both bacterial and fungal populations exhibited a non‐concentration‐dependent response to different concentrations of HDPE over time. In this study, we refer to the concentrations of 0.25%, 0.5%, 0.75%, and 1.0% as HT1, HT2, HT3, and HT4, respectively. Initial MP application significantly reduced bacterial colony counts for HT1, HT2, and HT4, while HT3 showed no significant change. On the 60th day, HT1 and HT3 exhibited a higher bacterial colony count compared to the control. On the other hand, fungal populations increased to maximum on day 1 but displayed no distinct time‐dependent trend from days 15 to 60. Furthermore, enzyme activities decreased with increasing concentrations of MPs over an extended period. Molecular docking studies suggest that HDPE can hinder enzyme activity by forming hydrogen bonds with enzymes. The C/N ratio was found to be significantly higher in MP‐treated soils on the 60th day relative to control, suggesting relatively slower degradation of carbon compounds in the MP‐treated soils.