The macro fungi of the genus Agaricus have so far recorded many bioactive compounds with antibacterial properties as a product of their metabolism. However, in Ecuador, A. pampeanus or “Kallamba de finados” is a species studied for its ethnomycological uses and as part of the fungal biodiversity of the country. In this study, the antibacterial activity of the ethanolic extract of A. pampeanus was evaluated on four ATCC strains and the bioactive compounds present in the extract were identified. Initially, an ethanol extract of A. pampeanus was performed with the use of a Soxhlet extractor. To identify the extracted compounds, a gas chromatograph coupled to a mass spectrophotometer was used. With the mother extract, four solutions of the A. pampeanus ethanol extract (APE) were made at different concentrations, classified in APE1 (50.32 mg/mL), APE2 (40.24 mg/mL), APE3 (30.16 mg/mL), APE4 (20.12 mg/mL), which were subsequently used in the analysis of antibacterial activity, using the Kirby-Bauer technique. Six chemical compounds were identified; among them Eugenol was found as one of the main compounds. Out of the four concentrations, the A. pampeanus ethanol extract (APE1) was the most effective against Enterococcus faecalis, Staphylococcus aureus and Pseudomonas aeruginosa and categorized as sensitive, while Escherichia coli did not present any activity and was cataloged as resistant. The APE4 was not effective in any case. The antibacterial effect of APE was verified. Furthermore, A. pampeanus is more effective against Gram positive bacteria.