The instantaneous response of a soil microbial community to a chemical stressor (Mentha spicata essential oil) was studied post acclimation to the same chemical treatment at lower exposure. Acclimation involved the repeated addition of small amounts of the essential oil weekly for a period of 1 month, while for the stress treatment, a 10-fold exposure level was introduced. We also tested the role of arbuscular mycorrhizal fungus (AMF) in the reponse of microbial community to the same stress exposure by pre-inoculating plant roots in the soil with the AMF Rhizophagus irregularis. Three days after stress exposure, the structure of the soil microbial community was investigated in addition to the activities of six soil enzymes mainly related to the N-cycle. The two preselected AMF inoculation and acclimation soil microbial communities responded differently to the subsequent stress. Acclimation enhanced the biomass of G+ bacteria, fungi and microeukaryotes, showing a priming effect of a low-intensity stimulus when applied repeatedly, while AMF inoculation decreased the biomass of these microbial groups. The relative changes in microbial biomasses in jointly pretreated samples were not different from the control, suggesting opposing effects of the two pretreatments. On the contrary, the jointly pretreated samples responded to stress exposure by exhibiting increased activity of asparaginase and glutaminase and reduced activity of arylamidase. Finally, the relationship between enzyme activities and certain microbial ratios denotes that specific activities depended on the relative abundance of specific functional groups (e.g., G+ or G−) rather than on their biomass per se.