Arthritis, a progressive inflammatory disorder, significantly impacts joint health and quality of life. Despite advancements in medical treatments, there remains a growing interest in traditional medicine systems for their holistic approaches and reduced risk of side effects. This study evaluates the anti-inflammatory and therapeutic potential of an Unani formulation comprising Anthemis nobilis Linn, Eucalyptus globulus, Ferula assa-foetida, Hyoscyamus niger, Styrax benzoin, and Sesamum indicum. Phytochemical screening revealed the presence of key bioactive compounds, including total phenolic content (TPC) and total flavonoid content (TFC), which contribute to their therapeutic properties. Antioxidant assays, such as DPPH, ABTS, FRAP, and NO, were performed using aqueous plant extracts to evaluate their free radical scavenging activity. Among the extracts, Ferula assa-foetida exhibited the highest antioxidant activity in both DPPH and ABTS assays, while Eucalyptus globulus showed the highest activity in the FRAP assay. For NO scavenging activity, Sesamum indicum demonstrated the strongest antioxidant potential. Acute toxicity testing was conducted on rats to evaluate the safety of these plants. Results indicated no toxic effects on the skin or other observed parameters, confirming their safety. Cytokine profiling studies revealed significant downregulation of key pro-inflammatory markers, including TNF-α and IL-6, highlighting the formulation’s potential to modulate inflammatory pathways effectively. These findings suggest that the selected plants can mitigate oxidative stress, reduce inflammation, and support joint health, providing a natural and effective alternative for arthritis management. The study underscores the potential of these medicinal plants as accessible, sustainable therapeutic options that integrate traditional practices with scientific validation.