Basidiomycete fungi have been extensively studied due to the wide variety of their bioproducts. The indiscriminate use of antibiotics is one of the main causes for the development of resistant bacterial strains, which do not respond to most of the available antimicrobials, being a great threat to human health. It is necessary to search for new effective therapeutic agents. Thus, the present work aims to verify the antibacterial activity of the aqueous extract of the mushroom Macrocybe titans as well as verify its bactericidal and/or bacteriostatic effects on ATCC strains of the pathogenic bacteria Bacillus cereus 10876, Salmonella Typhimurium 14028, Salmonella Enteritidis 13076, Listeria monocytogenes 19111, Escherichia coli 25922, E. coli O157:H7 43888, Pseudomonas aeruginosa 27853, Staphylococcus aureus 6538, B. subtillis subspecie Spizizenii 6633 and Klebsiella pneumoniae 13883. The fruiting body of the mushroom was dried in an oven, and then crushed in a knife mill until obtaining a fine powder. To obtain the aqueous fraction of the extract, the crushed material was mixed in autoclaved distilled water and kept at 4 ºC for 12 h. The antimicrobial susceptibility was evaluated by broth microdilution, according to M7-A6 (2003) and M02-A2 (2015) with some modifications for natural products. In microbiological analyzes it was possible to observe that the aqueous extract acted as a good antibacterial agent for both Gram-negative and Gram-positive bacteria. The minimum inhibitory concentration (MIC) for E. coli O157:H7 and K. pneumoniae bacteria was 7.81 mg/mL. As for E. coli, S. Typhimurium and B. cereus, it was 3.90 mg/mL. For P. aeruginosa, S. Enteritidis, L. monocytogenes, B. spizizenii and S. aureus was 1.95 mg/mL, being the best result. The values for the Minimum Bactericidal Concentration (MBC) test showed the same results as the MIC. Finally, the results allow us to affirm that the aqueous extract of M. titans presented a very promising behavior regarding its antibacterial action. The M. titans mushroom has no reports in the literature of its use as an antimicrobial agent. The continuation of the study is necessary to check if the antibacterial action found can be attributed to a specific compound or depends on some secondary compound produced by the mushroom.