There is a lack of information about the importance of fatty acid composition of the human sperm membranes and seminal plasma in the cryopreservation procedure. Our aims were to study the possible relationships between the fatty acid composition of human spermatozoa or seminal fluid before freezing, and the sperm quality, measured in terms of viability and motility, before and after freezing–thawing. A further objective of this study was to determine whether the antioxidant capacity (TAC) of the seminal plasma is related to fatty acid (FA) composition and to success of the cryopreservation process. Polyunsaturated fatty acids (PUFA), ω3 PUFAs and docosahexaenoic acid (DHA) in spermatozoa were significantly positively correlated with sperm viability and motility parameters before and after freezing. An inverse relationship was found for monounsaturated (MUFA), ratio ω6/ω3, ratio saturated saturated fatty acids/PUFA (SFA/PUFA) with the seminal parameters. Seminal plasma fatty acid composition was not related to viability. However, motility parameters before and after freezing were related to stearic acid (C18:0) and DHA. TAC in seminal plasma was directly related to PUFA, w3 and DHA. On the other hand, SFA, C22:0, C24:0 and MUFA in seminal plasma were inversely related to the antioxidant capacity. TAC was directly correlated with motion parameters after thawing, We described a significant correlation between the fatty acid composition of the human spermatozoa or seminal plasma and the sperm parameters of the samples after thawing. PUFA, W3 and specially DHA are directly correlated with sperm motility and viability after freezing/thawing, and MUFA was inversely correlated. This means that in the future the fatty acid composition could be used as a predictor of the capacity of cryopreservation. On the other hand, we could design further procedures to modify the lipid composition or/and antioxidant capacity of ejaculate to make it more resistant to the cryopreservation process.