The transition to a circular bioeconomy models promotes the exploration of natural, sustainable sources of bioactive compounds with potential therapeutic relevance. Polyphenols, especially anthocyanins, are known for their antioxidant and cytoprotective properties, making them promising agents in conditions associated with oxidative stress, such as male infertility. This study examines the effects of an anthocyanin-enriched fraction (EAF) from Callistemon citrinus (Lemon bottlebrush) flowers and its most rapresentative commercial anthocyanin, cyanidin 3-O-glucoside (cya-3-O), on Leydig cell viability, metabolic function, mitochondrial activity, and steroidogenic signaling. A green extraction protocol was employed, and the EAF was characterized via RP-HPLC-DAD-ESI-MS/MS, confirming an anthocyanin-rich profile (529.7 ± 5.40 mg equivalents of CyG/100 g DE composed of Cyanidin-3,5-O-diglucoside (56%), peonidin-3,5-O-diglucoside (34.2%), cyanidin-3-O-glucoside (7,2%), cyanidin-coumaroylglucoside-pyruvic acid (2,6%) . TM3 mouse Leydig cells were treated for 24 hours with EAF (0.01, 0.1, 1 μg/mL) and cya-3-O (0.5, 5, 50 μM). Cell viability, mitochondrial membrane potential, and intracellular ROS were assessed. EAF modulated cellular metabolic activity without inducing cytotoxicity, while cya-3-O led to a dose-dependent increase in LDH release, suggesting membrane damage at higher concentrations. No significant changes were detected in mitochondrial membrane potential or ROS levels. Western blot analysis was performed to assess mitochondrial respiratory complex activity, showing preliminary modulation in protein levels. In parallel, expression of key steroidogenic genes (STAR, CYP17A1, NRB02, SIRT1) was quantified via RT-qPCR, and protein activity of AC/PKA pathway was evaluated by Western blot. Results indicate that EAF and cya-3-O may modulate steroidogenesis through transcriptional and post-translational regulation. Ongoing analyses include ¹H-NMR-based exometabolomics and quantification of androstenedione hormone production. These findings suggest that EAF may influence Leydig cell function and bioenergetics, supporting their further investigation as potential modulators of male reproductive health. Additional studies are needed to fully elucidate the underlying molecular mechanisms and clinical potential.