The use of treated wastewater (WW) for irrigation is a common practice, especially in arid and semiarid agroecosystems. We aimed to evaluate the influence of long-term (up to 45 years) irrigation with WW on the soil microbial community structure, microbial activity and physicochemical properties, in comparison with soil irrigated with fresh water (FW), in a semiarid orange-tree orchard. Phospholipid fatty acid (PLFA) analysis was used to assess the shifts in the soil microbial community in response to the application of WW. Total organic carbon and available P increased significantly, by about 49% and 37%, respectively, due to WW irrigation. The urease, β-glucosidase, alkaline phosphatase and dehydrogenase activities and aggregate stability were higher in the soil irrigated with WW than in that irrigated with FW. The PLFA analysis showed a significant increase in bacterial abundance, particularly in G+ bacteria. The relative abundances of fungi, G− bacteria and actinobacteria were similar in the two soils. Principal components analysis of the PLFAs showed discrimination between the FW-irrigated soil and the WW-irrigated soil, which was enriched in actinobacterial PLFA 10Me18:0. The prolonged use of treated WW for irrigation in a semiarid agroecosystem promoted the establishment of a specific and persistent microbial community that was functionally more active.