Spirogyra is commonly found as accessible algae in freshwater areas all over the world. Some medical uses have been reported from this genus. Biological activities of Spirogyra singularis were investigated employing eight in vitro assays. The extract showed different antioxidant activity. IC50 for DPPH radical-scavenging was 4.71 ± 0.11 µg mL−1. The extract showed very strong nitric oxide-scavenging activity with IC50 = 77.3 ± 2.07 µg mL−1, but its Fe2+ chelating ability was weak. The extract also exhibited high antioxidant activity in hemoglobin-induced linoleic acid peroxidation tests and scavenging of hydrogen peroxide. The extract also showed good antihemolytic activity. The total amount of phenolic content in the extract was determined as gallic acid equivalents, and total flavonoid content was calculated as quercetin equivalents. Biological activities may be attributed, at least in part, to the presence of phenol and flavonoid contents in the extract.