Many variables are related to extra virgin olive oil (EVOO) quality and, therefore, to its polyphenols content and fatty acids composition including the steps that lead to EVOO extraction, considering that it is mainly contained in the vacuoles of the fruit mesocarp. The main steps of EVOO production are: harvest, crushing, malaxation, centrifugation, storage and filtration. Considering the olive oil extraction procedures, many studies have been conducted in recent years on oil mill plant and processes for improving EVOO quality. Malaxer is the most studied machine among all; it is responsible for malaxation, which represents a very important and critical step in the EVOO extraction process. Many studies focus on the control of oxygen in the malaxer headspace, with the aim of determining its influence on EVOO quality evaluating volatile and phenolic components. Considering these factors, the main objective of the present study was to evaluate the influence of the malaxer headspace oxygen concentration on EVOO fatty acids composition from cv. Nocellara del Belice olives. The results show that oxygen content in the malaxer headspace, in different time-points and concentrations during the process, influences EVOO fatty acids composition.