This research focuses on using spent coffee grounds as a source of caffeine by ultrasound-assisted extraction technique. Two types of ultrasound techniques (ultrasonic bath and ultrasonic probe) were studied to extract caffeine. The effect of the sonication type sonication power, extraction time, and extraction temperature on the extraction efficiency was investigated. The results demonstrated that extraction by an ultrasonic probe is superior to ultrasonic bath extraction. The highest caffeine recovery was obtained at 55OC by using an ultrasound bath where caffeine concentration was 795.74mg /L after 60 min. The bioactivity of extracted caffeine was also studied and compared with the bioactive of standard caffeine against candida albicance yeast. The results showed that the minimum inhibition concentration for natural caffeine was 100 mg/L which is half of the minimum inhibition concentration of standard caffeine.