Application of winery wastewaters to soils for irrigation of various crops or landscapes is a common practice in the wine industry. In this study, we sought to investigate the effects of this practice, by comparing the physicochemical and microbiological soil properties in paired sites that differed in having had a history of winery waste application or not. We also compared the effects of a single application of untreated winery wastewater, to application of treated winery wastewater (sequencing batch reactor) and pure water to eliminate the effects of wetting alone. Long-term application of winery wastes was found to have significant impacts on soil microbial community structure, as determined by phospholipid fatty acid analysis, as well as on many physicochemical properties including pH, EC, and cation concentrations. (13)C NMR revealed only slight differences in the nature of the carbon present at each of the paired sites. A single application of untreated winery wastewater was shown to have significant impacts upon soil respiration, nitrogen cycling and microbial community structure, but the treated wastewater application showed no significant differences to wetting alone. Results are discussed in the context of sustainable winery wastewater disposal.