The long-term effects of the olive mill wastewater (OMWW) spreading on soil chemical properties, microbial community, and olive tree parameters have been far poorly investigated. Therefore, this study aims to evaluate the effect of OMWW application on an olive orchard, and samples were collected at two different depths (0–20 cm and 20–40 cm) and 14 days, one year and two years from the end of the OMWW spreading on soil chemical characteristics and soil microbial structures. Variations of soil chemical parameters (pH, salinity, available P, and water-extractable organic C) were observed particularly at 14 days after spreading at both depths. All these parameters reached similar values to the soil after two years, except for available phosphorus. Firmicutes, Proteobacteria, and Actinobacteria are the most abundant phyla: only Firmicutes were negatively affected by the OMWW spreading after 14 days, suggesting that Gram-positive bacteria were probably negatively influenced by the addition of OMWW. The abundance of bacterial taxa in the soil is restored along time, except for the decrease of Firmicutes. This evidence suggests that the OMWW spreading in the long term does not affect the endemic soil bacterial community of the olive grove, as well as leaf net photosynthesis, the olive tree vegetative activity, yield, and fruits characteristics.