Crassostrea gigas is a sentinel species along the Italian coast. In mussels, the levels of oxidative stress biomarkers can be modulated by several environmental pollutants or pathogens and also fluctuate in response to reproductive stages and seasonal changes. In this study, adult Crassostrea gigas were sampled during summer and autumn from two lagoon and two offshore sites along the Adriatic coast of Italy in order to investigate the influence of seasonality on oxidative stress biomarkers. Trace elements load of Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Se and Zn suggests low contamination for lagoon and offshore sites. Levels of total glutathione, superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase and glutathione S-transferase were analyzed in digestive gland and gills of the Pacific oysters in June, July, September and October. OsHV-1 and Vibrio aestuarianus were detected in lagoon sites, but both pathogens did not affect the biomarkers levels in both tissues. Although several biological responses were found different among the four sites in the same month, principal component analysis revealed similar trend in biomarkers levels between sites during the whole sampling period. On the other hand, a different biochemical pattern through the months emerged, suggesting that the level of oxidative stress biomarkers in both tissues may be related to seasonal progress and biological cycle of oysters sampled from the two lagoons and offshore sites along the Italian coasts of the Mediterranean Sea.