Inflammation is an essential immune defense mechanism that protects the body from infections and tissue injury; however, when dysregulated or persistent, it becomes a pathological driver of chronic diseases. It is an essential immune response; however, its chronic activation contributes to the development of several disorders, including arthritis, cardiovascular diseases, and certain types of cancer. Natural plant extracts, enriched with bioactive compounds, have long been employed in traditional medicine to alleviate inflammation by modulating immune pathways. Mandragora autumnalis (MAE), traditionally recognized for its anticancer, antioxidant, and antimicrobial properties, is rich in phenols, flavonoids, and alkaloids; however, its anti-inflammatory mechanisms remain poorly understood. This study investigated the effects of ethanolic MAE extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Treatment with MAE significantly suppressed the expression of pro-inflammatory mediators, including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Furthermore, MAE reduced macrophage migration and regulated critical inflammatory signaling pathways, notably NF-κB, STAT-3, and MAPKs. Beyond its immunomodulatory activity, MAE exhibited broad-spectrum antimicrobial effects against diverse pathogenic bacterial and fungal strains. Collectively, these findings suggest that MAE possesses dual anti-inflammatory and antimicrobial properties, underscoring its potential as a natural therapeutic agent. Further studies are warranted to elucidate its molecular mechanisms and to explore its possible applications in the prevention and management of inflammation-associated disorders and infectious diseases.