The objectives of this work were to obtain steviol glycosides of S. rebaudiana leaves, possessing natural and noncaloric sweetener properties, using subcritical water extraction; to assess optimum extraction conditions; to determine biological activities of Stevia extracts and to characterize the raffinate phase. A Box-"Bhenken" statistical design was used to evaluate the effects of various values of temperature (100-150 degrees C), time (30-60 min) and flow rate (2-6 ml/min) at a pressure of 230 bar applying a solid/liquid ratio of 1:10 (m:v). The most effective parameter was temperature (p < 0.005). Optimum extraction conditions were elicited as 125 degrees C, 45 min, 4 ml/min flow rate which yielded 38.67 mg/g stevioside and 35.68 mg/g rebaudioside A. The total phenolic, flavonoid contents and DPPH free radical scavenging activity were found as 48.63 mg gallic acid/g extract, 29.81 mg quercetin/g extract and 92.50%, respectively. After extraction, total chlorophyll, carotenoid contents and dietary fibers were quantified as 31.91 mg/100g, 5.71 mg/100g and 4.98% in the raffinate phase. Hence, both extract and raffinate phases of S. rebaudiana leaves can be utilized as sources of natural sweeteners, fibers and coloring agents in the industry. (C) 2014 Elsevier B.V. All rights reserved.