Azoxystrobin has been largely applied as a kind of new strobilurin fungicide in recent years. Knowledge about the impact of the fungicide on soil biochemical properties is critical for understanding its effect on soil health. The effects of different doses of azoxystrobin (0, 0.1, 1, 10 mg·kg^-1) on cultivable soil microbial population, soil enzymatic activities, and soil respiration in Cambisol were investigated under laboratory incubation. These soil properties were determined after treatments of 7, 14, 21, and 28 days. The results indicated that azoxystrobin applications inhibited soil respiration, the populations of bacteria and actinomycetes, and dehydrogenase activity. However, catalase activity was activated as an effect of azoxystrobin application. Meanwhile, the treatment had no significant impact on fungi population and protease activity compared with the control. Urease activity was obviously inhibited with the azoxystrobin-treated soil in the first 14 days, but recovered close to the control levels on day 28. In general, azoxystrobin could cause certain negative effects on soil microbes in the acid soil with low organic matter.