Background - There has been an upsurge of interest in mushrooms, such as Pleurotus species, as an important source of bioactive compounds. Mycelia-submerged culture represents a promising approach to search new safe and healthy myco-products with standardized quality in addition to mushroom fruiting bodies. Methods - The study examined the in vitro antimicrobial, antitumor and antioxidant activities of a hot-water extract from Pleurotus sp. mycelium. The antimicrobial activity was screened through the activation of the microbial autolytic system of four bacteria and four yeast strains. The anti-proliferative effects on NB4 human leukemia cells were measured by flow-cytometry analyses. The antioxidant activity was investigated by the scavenging of DPPH and ABTS radicals, the reducing power and the inhibition of lipid peroxidation. Results and discussion - The extract activated the microbial autolytic system of eight strains: seven autolyzing strains with intensity values (Is) ranging from 2.7% in Candida sp. to 36.1% in Saccharomyces cerevisiae. Thus, the microbial autolytic system of the strains tested (including Gram-positive and Gram-negative bacteria as well as yeasts) could be activated in vitro by mycelial extract. Pleurotus extract reduced the viability of NB4 leukemia cells, particularly at the concentration of 200 μg/mL to 82% compared to control cells, and induced apoptosis demonstrated by an increase in annexin V-FITC+ cells (25% at 200 μg/mL). At 10 mg/mL, the extract showed the most potent scavenging effects for DPPH and ABTS radicals (96% and 55%, respectively) and the inhibition of lipid peroxidation (52%). The mushroom extract at 5 mg/mL manifested reducing power of 1.105. Although carbohydrates (76.8%, w/w) appear to be the most important bioactive compounds, secondary metabolites, like phenolics, would also contribute to the antioxidant, antimicrobial and anti-proliferative activities. Conclusions - The hot-water extract obtained from Pleurotus mycelium, in light of its in vitro antimicrobial, antitumor and antioxidant effects could be considered as a good candidate for developing nutraceuticals and for designing innovative myco-therapeutics and phytocosmetics applications.