This study investigated the contamination, composition, and functional properties of Wolffia globosa from northern Thailand. The results showed that the heavy metal content of dried W. globosa complied with Thai regulations, ensuring its safety. Its proximate analysis revealed high protein levels with lysine, leucine, and phenylalanine as the principal essential amino acids. The protein was effectively extracted using the alkaline extraction method, followed by precipitation induced by acid or heat. The precipitates and supernatants resulting from various acid- or heat-induced protein precipitation were obtained. The highest protein content was found in the pH 3 precipitate (51.15 ± 6.71%). In contrast, the pH 5 supernatant exhibited the most potent antioxidant activities (2.22 ± 0.05 mmol Trolox/mg and 4.55 ± 0.18 mmol Fe²⁺/mg), as determined by ABTS and FRAP assays, respectively. Additionally, a strong correlation was observed between phenolic content and antioxidant activity. Both supernatant and precipitate protein extracts from W. globosa exhibited no cytotoxicity in THP-1 cells and displayed anti-inflammatory effects by decreasing the production of IL-1β and IL-6. They also downregulated phospho-NF-κB, phospho-IκB-α, and COX-2, consistent with reduced NF-κB pathway activation. These findings position W. globosa as a promising, sustainable plant-based protein with bioactive and functional properties, making it a viable candidate for functional food formulations that enhance dietary health and add value to local agricultural resources.