The antibacterial and antifungal activities of the acetone extract from Paullinia cupana var. sorbilis Mart. (Sapindaceae) seeds, commonly called guarana, were assessed against selected bacterial and fungal strains. We tested the extract against both standard American Type Culture Collection (ATCC) and clinically isolated (CI) bacterial strains and three fungal strains. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for bacteria and MIC and minimum fungicidal concentration for fungi were determined. The extract showed an activity against the nine bacterial strains tested, both CI and ATCC strains (MIC comprised between 32 and 128 μm/mL and MBC between 128 and 512 μm/mL), showing a significant antibacterial effect against both Gram-negative and Gram-positive bacteria. Also, the tested fungi were sensitive to the extract (MIC between 125 and 250 μm/mL). The contemporaneous presence of different bioactivities in the extract from guarana suggests this plant as a source of bioactive substances.