Shiitake mushroom contains several therapeutic actions such as antioxidant and antimicrobial properties, carried by the diversity of its components. In the present work, extracts from shiitake mushroom were obtained using different extraction techniques: high-pressure operations and low-pressure methods. The high-pressure technique was applied to obtain shiitake extracts using pure CO2 and CO2 with co-solvent in pressures up to 30 MPa. Organic solvents such as n-hexane, ethyl acetate and dichloromethane were furthermore used to produce the shiitake extracts in low-pressure extraction process. The different extraction procedures were evaluated for antioxidant activity by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) essays and the results compared with data from Folin–Denis method, used to measure the total phenolic content. Antimicrobial activities of the extracts were also subjected to preliminary screening against four strains of bacteria and one fungal strain using agar dilution method. The results indicate that the fractions obtained with CO2 using ethanol as co-solvent, at 40 °C, 20 MPa and 15% EtOH, and for dichloromethane in low-pressure technique had similar antioxidant activities. Furthermore, only the supercritical fluid extracts had antimicrobial activity against Micrococcus luteus and Bacillus cereus. The shiitake extraction yields were up to 3.81% w/w and up to 1.01% w/w for supercritical fluid extraction with ethanol as co-solvent and with pure CO2, respectively, while the low-pressure extraction indicates yields up to 1.25% w/w for n-hexane as solvent.