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The limited availability of fresh water caused by climate change and global population increase has made research necessary into alternative water sources which are sustainable but of lower quality, in order to meet the demands of agriculture in arid and semi-arid areas. However, there are concerns that irrigation with treated wastewater (TWW) could lead to a deterioration of soil health and a decline in plant yield. The objective of the field study was to determine the effects of medium-term (3-year) irrigation with secondary TWW, fresh water (FW) and a mix of 1:1 TWW/FW (HW) on various microbiological and biochemical soil properties of a Typic Xerofluvent soil and on maize (Zea mays L.) productivity in Mediterranean climatic conditions. In line with this aim, microbial biomass C (MBC), basal soil respiration (BSR) and nitrogen mineralization (N-min), and the activities of the enzymes alkaline phosphatase (ALKPA), dehydrogenase (DHG), beta-glucosidase (GLU), aryl sulphatase (ArSA) and urease (UA) were determined. In addition, the grain yield of maize was measured. Taking three-year averages into account, it was found that although all biological dependent variables measured in connection to the application of TWW had been affected in a range of 2% to -11% (mean 3%), the differences created by the use of FW did not reach the level of statistical significance. With the application of HW, a significant and more severe reduction of -19% (P<0.05) was found. The application of TWW increased only ALKPA and ArSA activity, by 4% and 2%, respectively, and this result was found not to be significant. Even though the effect of TWW on soil health was not found to be significant, a negative effect was found on the grain yield of maize plants (P<0.05) at a level of 17% (1.68 t ha(-1)). The fact that no significant effect was found of the TWW used in this study conducted under Mediterranean climatic conditions on soil health is an indicator of its potential long-term use with different crops and different irrigation techniques. Furthermore, it has been proved that the re-use of treated wastewater, which has an effect close to that of fresh water, can be an important part of the integrated management of water resources, and is a reliable alternative water source, which can provide an effective solution in conditions of water shortage.