BACKGROUND The aim of this study is to investigate in vitro antioxidant, antimicrobial and anticancer activity of the acetone extracts of the lichens Cladonia furcata, Lecanora atra and Lecanora muralis. METHODS Antioxidant activity was evaluated by five separate methods: free radical scavenging, superoxide anion radical scavenging, reducing power, determination of total phenolic compounds and determination of total flavonoid content. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method against six species of bacteria and ten species of fungi. Anticancer activity was tested against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines using MTT method. RESULTS Of the lichens tested, Lecanora atra had largest free radical scavenging activity (94.7% inhibition), which was greater than the standard antioxidants. Moreover, the tested extracts had effective reducing power and superoxide anion radical scavenging. The strong relationships between total phenolic and flavonoid contents and the antioxidant effect of tested extracts were observed. Extract of Cladonia furcata was the most active antimicrobial agent with minimum inhibitory concentration values ranging from 0.78 to 25 mg/mL. All extracts were found to be strong anticancer activity toward both cell lines with IC(50) values ranging from 8.51 to 40.22 μg/mL. CONCLUSIONS The present study shows that tested lichen extracts demonstrated a strong antioxidant, antimicrobial and anticancer effects. That suggest that lichens may be used as as possible natural antioxidant, antimicrobial and anticancer agents to control various human, animal and plant diseases.