Phospholipase A2 (PLA2) is involved in eicosanoid release, and F2‐isoprostanes (F2‐IsoPs), as free radical‐generated eicosanoids released by PLA2, are indicators of oxidative stress in different human conditions. This study investigated the interplay between cytosolic PLA2 (cPLA2), F2‐IsoPs and sperm features in male infertility, when the involvement of oxidative stress has been reported. Semen evaluation was performed following WHO guidelines, sperm ultrastructure was detected by transmission electron microscopy indicating a fertility index, and the percentages of sperm immaturity, apoptosis and necrosis. In sperm cells and seminal plasma, cPLA2 levels were determined by immunological method, whereas F2‐IsoPs by mass spectrometry. Sperm concentration, morphology, vitality and fertility index values were significantly lower in infertile groups compared with fertile men. An increase in sperm apoptosis and necrosis (p <.01), apoptosis (p <.01) and immaturity (p <.001) was detected in leucocytospermia, idiopathic infertility and varicocele, respectively. Seminal cPLA2 showed the highest value in varicocele group (p <.05), whereas seminal F2‐IsoPs increased in varicocele (p <.001) and leucocytospermia (p <.05) groups. In the whole population, F2‐IsoP and cPLA2 levels were positively correlated (p <.05). On the contrary, F2‐IsoPs and cPLA2 were not significantly different when investigated in sperm cells. Our data indicate that fatty acid oxidation/metabolism plays a role in different male reproductive pathological conditions. [ABSTRACT FROM AUTHOR]