tIn this work, extracts of chamomile (Matricaria chamomilla L.) ligulate flowers obtained by Soxhlet,microwave-assisted, ultrasound-assisted and subcritical water extraction were compared in respectto their bioactivity, more specifically antioxidant, antimicrobial and cytotoxic activity. In all obtainedextracts, the content of total phenols and flavonoids was determined, indicating significant total extrac-tion yields (49.70%) and total phenol content (151.45 mg ECA/ml) in water extracts obtained undermoderate subcritical conditions. By applying DPPH and Reducing Power Test subcritical water extractionshowed superior free-radical scavenging ability in comparison to other investigated extraction tech-niques. Microbial properties of extracts were examined using eight selected indicator strains and for allextracts minimum inhibitory concentrations were in the range between 19.53 and 312.50 µ�g/ml. Subcriti-cal water extracts showed the highest activity against Escherichia coli (MIC = 39.10 �µg/ml) and Aspergillusniger (MIC = 39.10 µ�g/ml). Cytotoxic effects of extracts were examined on human rhabdomyosarcomacells (RD), cell line derived from human cervix carcinoma Hep2c (HeLa) and cell line derived from murinefibroblast (L2OB). On all three tested cell lines, subcritical water extracts demonstrated much better anti-tumor properties in comparison to extracts obtained by other techniques, with IC50values for Hep2c, RDand L2OB cells 30.54, 20.54 and 19.65 µ�g/ml, respectively.