The aim of this study is to assess and optimize Cytisus villosus Pourr. biological activities using response surface methodology (RSM). Antioxidant (DPPH, FRAP, ABTS and molybdenum assays) and anti microbial (antifungal and antibacterial) activities of different C. villosus extracts have been determined. Central composite design (CCD) was used to investigate the effect of temperature, extraction time, Liquid/Solid ratio, and solvent concentration on total phenolic yield (TPY), AntiStaphylococcus aureus (ASA), Anti-Escherichia coli (AEC) and DPPH radical scavenging activities (DRS). The used model has a good fitting with all dependent parameters (p˂0.01). By applying optimal extraction conditions, maximum antioxidant and antibacterial activities have been found (20 mm, 12mm, and 87.01% for ASA, AEC and DRS, respectively). The results of HPLC/diode-array detector (DAD) analysis of C. villosus methanolic extract showed that rutin, quercetin and catechin are the most abundant compounds, which can explain the remarkable antioxidant and antimicrobial activities showed by this extract.