Despite the technical and scientific advances in the health field, infectious diseases continue to be one of the main causes of death worldwide, with microbial resistance being the main aggravating factor of this problem. Medicinal plants have a very rich composition of phytochemicals that can be structurally optimized and processed into new drugs. The objective of this study was to investigate the antibacterial potential of the extract and fractions prepared from the stem bark and leaves of Caryocar brasiliense Cambess using in vitro assays. Plant phytochemical screening was performed using precipitation and staining methods and antibacterial activity using disk diffusion and broth microdilution method against standardized strains of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The results of phytochemical screening revealed the presence of flavonoids, free steroids, free pentacyclic triterpenoids, saponin and alkaloids. In this study, the crude extracts and fractions of C. brasiliense showed inhibitory effect against S. aureus, K. pneumomiae and P. aeruginosa with inhibition halos ranging from 7.5±1.40 to 18.3±6.06 mm, being S. aureus more sensitive to disc diffusion tests. By broth microdilution the extracts were most effective against K. pnuemoniae (Minimum Inhibitory Concentration (MIC) = 125-1000 μg/mL) followed by S. aureus (MIC = 250-1000 μg/mL). The extracts were inactive against E. coli. Based on the results, C. brasiliense extracts may become alternative agents for the control of infections associated with these bacteria after toxicity studies and in vivo testing. © 2021 by the authors. Esta revista oferece acesso livre imediato ao seu conteúdo, seguindo o princípio de que disponibilizar gratuitamente o conhecimento científico ao público proporciona maior democratização mundial do conhecimento. Este manuscrito é distribuído nos termos da licença Creative Commons - Atribuição - NãoComercial 4.0 Internacional (https://creativecommons.org/licenses/by-nc/4.0/legalcode), que permite reproduzir e compartilhar o material licenciado, no todo ou em parte, somente para fim não comercial; e produzir, reproduzir, e compartilhar material adaptado somente para fim não comercial.