The freshwater green algae, S. neglecta, has been commonly used as a Northern Thai local food due to the fact that it is composed of several nutritional components. The aim of the present study was to investigate the phytochemical properties and biological activities of an S. neglecta water extract for application as a functional food or pharmaceutical ingredient. The phenolic and flavonoid contents of S. neglecta and its biological activities, including anti-oxidant, pancreatic cholesterol esterase, anti-inflammatory, cytotoxic, and antibacterial activities, were investigated in vitro. The results showed that the S. neglecta extract contained 157.92 mg GAE/g extract of phenolics and 10.10 mg CE/g extract of flavonoids. The radical scavenging activity of the S. neglecta extract exhibited an IC50 of 7.46±1.17 µg/mL from DPPH assay and 224.20±1.06 µg/mL from ABTS assay. Meanwhile, trolox exhibited an IC50 of 7.03±1.08 µg/mL from DPPH assay and 5.24±1.10 µg/mL from ATBS assay. Interestingly, the S. neglecta extract at 10 mg/mL inhibited pancreatic cholesterol esterase activity by 60 %. Moreover, the release of TNF-α from macrophages was strongly reduced by incubation with the S. neglecta extract in a dose-dependent manner, and all of the study concentrations were non-toxic to primary fibroblast cells. In addition, the S. neglecta extract inhibited some gram-positive and -negative bacteria. In summary, S. neglecta extract could possibly be used as a potential functional food or pharmaceutical ingredient, for which further studies are required.