Background: Polycystic ovary syndrome (PCOS) is characterized by altered follicular development and metabolic dysfunction, frequently exacerbated by obesity. The follicular fluid (FF) microenvironment plays a critical role in supporting oocyte maturation and granulosa cell function; however, the extent to which FF from women with PCOS and obesity is associated with alterations in granulosa cell metabolism remains unclear. This study aimed to evaluate how does the FF from women with PCOS and/or obesity shapes granulosa cell glycolytic and mitochondrial activity. Results: FF from women with PCOS showed significantly increased concentrations of total testosterone and -androstenedione compared with controls, irrespective of BMI (p < 0.05 and p < 0.01, respectively). Exposure of human granulosa cell line (HGrC1) to FF from women with PCOS and obesity was associated with a marked reduction in glycolytic capacity (p < 0.05) and decreased mRNA expression of key glycolytic regulators, including GLUT1, HK2 and LDHA (p < 0.05). Mitochondrial function was also altered, as evidenced by reduced maximal respiration and mitochondrial membrane potential (p < 0.05), while reactive oxygen species levels remained unchanged. Metabolomic profiling revealed elevated glucose concentrations in FF from women with PCOS and obesity compared with normal-weight controls, consistent with potential alterations in glucose metabolism within the follicular environment. Conclusion: Granulosa cells depict metabolic dysregulation, with reduced glycolytic activity and impaired mitochondrial function, when exposed to FF from women with PCOS, which is further exacerbated in the presence of obesity. These findings from a pilot hypothesis-generating study suggest that the intrafollicular environment may be associated with granulosa cell metabolic disturbances, which warrant mechanistic studies to establish causality and elucidate the downstream consequences for follicular maturation and ovulatory function.