Several species of the genus Aspidosperma are used in folk medicine as a potential agent against malaria, leishmaniasis, antimicrobial and inflammatory process. For this study we selected the species Aspidosperma tomentosum, A. macrocarpum and A. pyrifolium commonly known as "peroba". The aim of this study was to evaluate the antimicrobial activity of extracts and fractions of these species against strains of Gram positive and Gram negative bacteria and yeasts. We used ethanol extracts of root bark, stem bark, stem and root of A. tomentosum, A. macrocarpum were used ethanolic extracts of the twigs and stems, leaves, stem bark and stem, and the fractions obtained from crude extract of the stem. For the species A. pyrifolium were used crude ethanol extract of the fruit, flower, root, root bark, bark of the wood and timber, and the fractions of crude ethanol extract of the wood. The antimicrobial activity was observed on strains of Gram positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6623), Gram negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 15442) and Candida species (Candida albicans ATCC 10231, C. parapsilosis ATCC 22019 and C. tropicalis), by broth microdilution test for determining the minimum inhibitory concentration (MIC) and minimal bactericidal and fungicidal concentration (MBC/MFC). The extracts and fractions of A. macrocarpum showed no activity against the tested bacteria (MIC > 1000 μg/mL) and ethyl acetate fraction (MIC 250 μg/mL) and the alkaloidal fraction (MIC 500 μg/mL) of the stem showed weak activity for C. parapsilosis. The crude extract of root bark of A. tomentosum showed a weak antibacterial activity in S. aureus and B. subtilis (MIC 1000 and 500 μg/mL, respectively) and showed no activity against yeasts (MIC > 1000 μg/mL). For the species A. pyrifolium only the alkaloidal fraction of the wood showed a moderate activity with MIC of 125 and 250 μg/mL in S. aureus and B. subtilis, and the MBC was 1000 μg/mL for both bacteria. This fraction also showed a weak activity against C. parapsilosis and C. tropicalis (MIC 500 μg/mL). Although there are ethnopharmacological reports about the popular use of some species of Aspidosperma as antimicrobial, as this study found no significant activity for this action in the extracts and fractions of A. tomentosum, A. macrocarpum and A. pyrifolium against the tested microorganisms. Tests for verification of the action potential of these extracts on protozoa are being conducted.