Obesity and its associated metabolic disorders are significant global health challenges, necessitating novel therapeutic approaches. This study explores the anti-adipogenic and anti-dyslipidemic properties of 4655-EF, a novel phytopharmaceutical derived from Polyalthia longifolia, and explores the molecular pathways involved in its pharmacological activity. This phytopharmaceutical was prepared using a bioactivity-guided supercritical fluid extraction method suitable for large-scale production. The RP-UHPLC analysis of 4655-EF revealed that the percentage composition of its four biomarkers was 21.19 ± 1.21% (N-016-0014), 0.83 ± 0.02% (N-016-0015), 0.3 ± 0.02% (N-016-0016), and 35.09 ± 1.57% (N-016-0017). We examined the effects of 4655-EF on HFD-fed Syrian Golden Hamsters and HFD-fed C57BL/6 mice, models of dyslipidemia and obesity, respectively. In the hamsters, 4655-EF treatment reduced body weight and fat mass and improved lipid parameters. Similarly, in the mice, 4655-EF treatment resulted in reduced body weight and fat mass and improved dyslipidemia, glucose tolerance, and insulin sensitivity. Moreover, mechanistic study exploring the possible pathways responsible for its anti-adipogenic activity uncovered the downregulation of genes associated with adipogenesis, cholesterol metabolism, and PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathways using next-generation sequencing. Additionally, miRNA expression analysis indicated the activation of the anti-adipogenic Wnt/β-catenin pathway. These findings signify a multifaceted mechanism by which 4655-EF exerts its beneficial effects and highlight its potential as a promising phytopharmaceutical for addressing obesity and dyslipidemia, along with its industry-friendly method for large-scale production.