Imidacloprid is one of the most commonly used insecticides in agricultural practice, and its application poses a potential risk for soil microorganisms. The objective of this study was to assess whether changes in the structure of the soil microbial community after imidacloprid application at the field rate (FR, 1 mg/kg soil) and 10 times the FR (10x FR, 10 mg/kg soil) may also have an impact on biochemical and microbial soil functioning. The obtained data showed a negative effect by imidacloprid applied at the FR dosage for substrate-induced respiration (SIR), the number of total bacteria, dehydrogenase (DHA), both phosphatases (PHOS-H and PHOS-OH), and urease (URE) at the beginning of the experiment. In 10x FR treated soil, decreased activity of SIR, DHA, PHOS-OH and PHOS-H was observed over the experimental period. Nitrifying and N-2-fixing bacteria were the most sensitive to imidacloprid. The concentration of NO decreased in both imidacloprid-treated soils, whereas the concentration of NI-a in soil with 10x FR was higher than in the control. Analysis of the bacterial growth strategy revealed that imidacloprid affected the r- or K-type bacterial classes as indicated also by the decreased eco-physiological (EP) index. Imidacloprid affected the physiological state of culturable bacteria and caused a reduction in the rate of colony formation as well as a prolonged time for growth. Principal component analysis showed that imidacloprid application significantly shifted the measured parameters, and the application of imidacloprid may pose a potential risk to the biochemical and microbial activity of soils. (C) 2014 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.