Simple SummaryReproductive seasonality is a major factor affecting buffalo breeding. The rationale of this work derives from the hypothesis that the reduced cleavage and blastocyst rates observed during the non-breeding season could be due to a suboptimal follicular environment. The present study aimed to evaluate the influence of season on the lipid profile of the ovarian follicle in the Italian Mediterranean buffalo. For this purpose, abattoir-derived ovaries were collected during the breeding and non-breeding seasons, and the apolar phase of follicular components was analyzed. To our knowledge, this is the first report of seasonal variations in lipid content of the buffalo ovarian follicle, including follicular fluid, follicular and cumulus cells, and oocytes. The results undoubtedly demonstrated significant seasonal variations in the lipid profile, including triglycerides, cholesterol, and phospholipids, in the different biological matrices analyzed. Interestingly, an increased amount in the total level of non-esterified fatty acids in the follicular fluid was also observed during the non-breeding season. The results allow a better understanding of the physiology of the ovarian follicle in buffalo and unveil some causes of reduced oocyte competence during the non-reproductive season, laying the groundwork for further studies and corrective strategies.The reduced oocyte competence recorded during the non-breading season (NBS) is one of the key factors affecting the profitability of buffalo farming and limits the IVEP efficiency. The purpose of this experiment was to evaluate whether season influences the lipid content within the ovarian follicle in the Italian Mediterranean buffalo. Abattoir-derived ovaries were collected during the breeding season (BS) and the NBS, and different matrices (follicular fluid, oocytes, cumulus and follicular cells) were recovered. After the extraction of the apolar fraction, all samples were analyzed by H1 nuclear magnetic resonance and FF samples by gas chromatography–mass spectrometry. Seasonal differences in lipid composition were observed in all matrices. In particular, during the NBS, the triglyceride content was higher in the follicular fluid and in the oocytes but reduced in the follicular cells. Both cholesterol and phospholipids were reduced in the follicular fluid and follicular cells during the NBS. Furthermore, the total amount of non-esterified fatty acids was significantly increased in the follicular fluid. The seasonal variation in lipid profile of the follicle may, in part, account for the reduced buffalo oocyte competence during the NBS, due to the critical role played by lipids in regulating ovarian functions.