Three genetically identified and morphologically characterized strains (MesAQ2-C, MesAQ6-2 and MesFI2-3) of the culinary-medicinal ascomycete mushroom Morchella esculenta (L.) Pers. collected in central-north Italy have been studied for their antifungal and antibacterial activities. The obtained data showed that mycelium of M. esculenta possess variable antimicrobial activity against four test fungi (Chrysosporium keratinophilum, Microsporum gypseum, Trichophyton terrestre, Penicillium griseofulvum), as well as one Gram positive (Staphylococcus aureus) and three Gram negative (Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa) test bacteria potentially pathogenic for humans and animals. Up to 20.4% of inhibition of the average mycelial growth rate (GRavr) of test fungi in dual culture experiment was detected. The samples of cultural liquid (CL) and mycelial extract (ME) obtained by static cultivation of M. esculenta strains showed up to 13.9 and 23.0% of GRavr inhibition of test fungi, respectively. Similarly, the inhibition of the bacterial colonies by CL and ME samples was 34.1 and 32.3%, respectively in comparison with the control with streptomycin indicating almost equal secretion of both intra- and extracellular antimicrobial compounds by M. esculenta mycelium. As a producer of antimicrobial compounds among tested M. esculenta strains, MesAQ2-C was the most effective. It may be considered for further myco-pharmacological research to develop mushroom-based antimicrobial biotech products with biomedical significance.