Abstract Subcritical water extraction is gaining increased popularity in the production of natural extracts. Higher yields of target compounds, broader range of extracted compounds, its green character and safety make this extraction technique convenient for food, pharmaceutical and cosmetic industry. The rhizome of ginger (Zingiber officinale) has been used in many traditional medicines. Modern science also recognises and confirms its beneficial health effects including antioxidant, antimicrobial and antiviral activity. Subcritical water extracts of ginger (Zingiber officinale) rhizome were compared to extracts obtained by boiling water at atmospheric pressure in respect to antimicrobial and cytotoxic activity. Antimicrobial activity was evaluated against eight microbial strains two of which belong to fungal class. Cytotoxic activity was observed for three tumor cell lines derived from human cervix carcinoma, human rhabdomyosarcoma and murine fibroblast. Chemical composition of subcritical water extracts of ginger was determined by HPLC-ESI-TOF–MS.