Introduction: The use of wastewater for irrigation in arid and semi-arid regions of the world is increasing. This study aimed to evaluate the effects of wastewater on the microbial activity of irrigated soils using the enzymatic activity of soil microorganisms. Materials and Methods: In this study, for soil irrigation, the secondary effluent of the Isfahan municipal wastewater treatment plant was used. As a control, tap water that has no microbial load was also used. Soil samples were collected in two stages, before and immediately after irrigation. All samples were collected in sterile bags, transferred immediately to the laboratory for physicochemical and microbiological tests. Soil samples were analyzed for the amounts of enzymatic activity (Fluorescein Diacetate (FDA) and dehydrogenase), electrical conductivity (EC), oxidation-reduction potential (ORP), and pH. Results: The EC levels before and after irrigation with tap water was 231.2 and 260.63 µs.cm-1, respectively, which was significantly different from levels of wastewater-irrigated soil (P < 0.05). pH in the two types of used water before and after irrigation was 6-8 and 7-8, respectively. No significant difference was observed in the levels of FDA, dehydrogenase, ORP, and microbial population in samples irrigated with water and wastewaters (p > 0.05). It was found that there is a significant relationship between bacterial density and FDA (P < 0.05). Conclusion: The results of the study showed that irrigation with wastewater has no significant effect on the microbial activity of irrigated soil. Because of the short-term wastewater irrigation in the present study, however, further investigation is needed to evaluate the effect of long-term wastewater irrigation on the microbial and physicochemical quality of soil.