The emergence of new bacterial strains with multiple resistances may lead to the emergence of new hospitalacquired infections that result in high mortality. Research on new antiinfectives must therefore be continued and all possible strategies should be explored. Screening of plant extracts as well as secondary metabolites, aiming to highlight the presence of new compounds with antimicrobial activity is a potential source of more effective drugs. In this paper we report the antibacterial and hemolytic activities of the ethyl acetate fraction and compounds 1 (8-formyl-3,5-dihydroxy-7- methoxy-6-methylflavanone) and 2 (3,4,6-trihydroxy-2-methoxy-5-methylchalcone) isolated from Piper montealegreanum, which have not shown any previous biological study. The ethyl acetate fraction from Piper montealegreanum showed antibacterial activity against Gram positive (B. subtilis) and Gram negative (E. coli and P. aeruginosa) resistant to ampicillin at lower concentrations showing no cytotoxic effect against human erythrocytes (A, B and O), and is therefore a potential broad-spectrum antibacterial agent. Both the flavanone and the chalcone showed antibacterial activity against P. aeruginosa resistant to ampicillin, which means that they can be used as narrow–broad antibacterial agents.