In this study, phenolic profile of Allium scorodoprasum L. subsp. rotundum and its bioactivity in multiway approach including antioxidant, antimicrobial, antidiabetic, cytotoxic and antiinflamatory activities were investigated. The extraction of phenolic and flavonoid compounds from Allium scorodoprasum L. subsp. rotundum was optimized as a function of ethanol percentage, extraction temperature and extraction time by employing a response surface method. The optimal extraction conditions were found as 79 degrees C extraction temperature, 28% ethanol and 118 min duration of the extraction. Quercetin was determined as the major phenolic compound of the extract obtained under the optimal conditions and followed by vanillic, caffeic, 2,4 hydroxybenzoic and ellagic acid. In addition to high antioxidant capacity, the obtained extract showed antimicrobial activity, against different bacteria and molds. The concentrations of the extract obtained at the optimal conditions that inhibited 50% of Aspergillus oryzae and pancreatic alpha-amylases and alpha-glycosidase activities were calculated as 11.56, 14.35 and 19.35 mg extract/mL, respectively. The antiinflammatory effect of the obtained extract was also examined, and it was more inhibitory against xanthine oxidase activity than against lipoxygenase activity. The cytotoxicity of the optimized extract was observed against breast and bone cancer cells, and it showed significant cytotoxic activity especially on bone cancer cells. As a result, Allium scorodoprasum L. subsp. rotundum could serve as a promising source of natural bioactive compounds for application in food and non-food products.