Zanthoxylum rhetsa (Roxb.) DC. (Ma-Khwaen) is an economically important medicinal plant traditionally used for pain and inflammatory disorders. This study explored its phytochemical composition, biological activities, and potential for nano-spray product development. Methods: Ethanolic fruit extracts collected from five provinces in Northern Thailand were evaluated for extraction yield, total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, and anti-inflammatory activity. Extracts from Nan Province were further selected for DPPH radical scavenging, GC–MS characterization, nitric oxide (NO) inhibition assay in LPS stimulated RAW 264.7 macrophages, nano-spray formulation development, and stability assessment. Results: Considerable phytochemical variation was observed among geographical origins, with TPC and TFC ranging from 19.47 to 103.09 mg GAE/g extract and 28.57 to 400.72 mg QE/g extract, respectively. The Nan whole-fruit extract exhibited the highest extraction yield (20.81%), whereas the pericarp extract showed the strongest antioxidant activity (IC₅₀ = 111.78 μg/mL). GC–MS analysis revealed anti-inflammatory phytochemicals, predominantly α-terpineol, geranyl acetate, n-hexadecanoic acid, oleic acid, and linalool. The selected extract demonstrated potent anti-inflammatory activity (IC₅₀ = 6.95 ± 0.44 μg/mL), exceeding that of prednisolone, and was successfully incorporated into a physically stable nano-spray formulation with 60-day storage stability. Conclusions: These findings provide scientific validation for the traditional use of Ma-Khwaen and support its development as a stable nano-spray formulation for muscle pain relief and inflammation alleviation.