In the present study, we report for the first time the antioxidant and antimicrobial activities of Withania frutescens (L.) Pauquy roots and leaves. Total phenolic content was determined using the Folin-Ciocalteu method and antioxidant activity was evaluated by the DPPH free radical scavenging and reducing power methods. Antimicrobial activity tests were carried out against ten bacterial species involved in nosocomial infections and two opportunistic clinical yeast isolates. The ethyl acetate and n-butanol leaf fractions exhibited the highest DPPH radical scavenging activity with IC50 = 4.53 +/- 0.12 and 8.49 +/- 0.46 microg/mL, respectively. The n-butanol root fraction showed the greatest reducing power comparable with that of quercetin at 0.4 mg/mL. The dichloromethane leaf fraction exhibited the highest antibacterial activity against both Gram-positive and Gram-negative bacteria with MIC values ranging between 50 and 400 microg/mL, depending on the tested bacteria. However, none of the examined extracts exhibited anticandidal activity. The polyphenol and glycowithanolide constituents appeared to be responsible for the antioxidant capacity of W. frutescens, whereas the observed antimicrobial activity may be due to the presence of withanolides.