Olive oil mill wastewater (OOMW) is considered the most polluting fraction of olive processing residues, due to its high content in polyphenols. In this study it has been examined the possibility of using an ozone source as a strong oxidant agent, to lower the phenolic fraction of OOMW, allowing its use in agriculture. The OOMW, coming from a continuous 3-phase olive oil mill located in the province of Arezzo (Italy) was submitted to ozonation for 1, 3, and 8 hours, using an ozone generator. Total polyphenols, antioxidant activity, and the amount of tyrosol and hydroxytyrosol were determined on the derived samples. To measure the biotoxicity of the treated OOMW the germination test of radish seeds was used. The results of the chemical determinations highlighted the effect of dephenolyzation performed by ozonation of OOMW, with a significant decrease of antioxidant activity. Hydroxytyrosol was significantly lowered, depending on the duration of treatment, while tyrosol resulted less affected. The germination test showed that, with a 50% dilution of OOMW, the biotoxicity decreases as the ozone treatment increases.