The present study reported the optimization of extraction of phenolic and flavonoid compounds from the cherry stem and biological activities of the extract. The extraction parameters were optimized employing a response surface methodology, and they were determined as 79 degrees C, 35% (vol/vol) of ethanol percentage and 119 min. It was found that the extract obtained at the optimum conditions contained high amounts of sinapic acid and quercetin followed by caffeic and ferulic acid. It had a high antioxidant capacity and showed antimicrobial activities againstEscherichia coli,Enterococcus fecalis,Staphylococcus aureus,Aspergillus flavus,andAspergillus niger. The extract inhibited alpha-amylase, alpha-glucosidase, lipoxygenase, and xanthine oxidase enzymes which indicated that the extract had antidiabetic and anti-inflammatory effects. It also showed cytotoxic activities on the breast and bone cancer cells. The results showed that cherry stems might be potentially useful in food or nonfood applications and an important natural source for bioactive compounds. Practical applications The cherry stem extract showed the high antioxidant, antimicrobial, antidiabetic, and anti-inflammatory activities. It displayed cytotoxicity against MCF-7 and MG-63 cells. It can replace the chemical compounds in food and nonfood formulations to control the oxidative change and undesirable microbial activity and can reduce the blood glucose levels, inflammation and the risk of cancer, and it can meet the increasing consumer demand to the natural products.