In this study, antimicrobial, antioxidant and cytotoxic activities of Spartium junceum L. aromatic water (SJAW), obtained by vacuum process distillation, were investigated. Antimicrobial activity of SJAW was evaluated against fungal organisms, Gram-positive and Gram-negative bacteria using agar-well diffusion method. Moreover, the ability of SJAW to sensitize Staphyloccus aureus to antibiotics was also evaluated. The antioxidant activity was examined by 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay and the cytotoxic activity of SJAW main components was tested on melanoma RPMI 7932 and normal keratinocytes NCTC 2544 cell lines using the trypan blue method. The preparation did not show antibacterial activity against tested microorganisms and was unable, at tested concentrations/conditions, to sensitize Staphylococcus aureus to antibiotics clindamycin (2 μg) and tetracycline (30 μg). On the contrary SJAW possesses an appreciable antioxidant and cytotoxic activity suggesting its potential use for pharmaceutical applications.