Endophytic fungi were isolated from healthy leaves of Indigofera suffruticosa Miller, a medicinal plant found in Brazil which is used in folk medicine to treat various diseases. Among 65 endophytic fungi isolated, 18 fungi showed activity against at least one tested microorganism in preliminary screening, and the best results were obtained with Nigrospora sphaerica (URM-6060) and Pestalotiopsis maculans (URM-6061). After fermentation in liquid media and in semisolid media, only N. sphaerica demonstrated antibacterial activity (in Potato Dextrose Broth-PDB and in semisolid rice culture medium). In the next step, a methanolic extract from rice culture medium (NsME) and an ethyl acetate extract (NsEAE) from the supernatant of PDB were prepared and both exhibited antimicrobial activity against Gram-negative and Gram-positive bacteria. The best result was observed against Staphylococcus aureus, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 1.56 mg/mL and 6.25 mg/mL, respectively, for NsME and MIC and MBC values of 0.39 mg/mL and 3.12 mg/mL, respectively, for NsEAE. This study is the first report about the antimicrobial activity of endophytic fungi residing in I. suffruticosa leaves, in which the fungus N. sphaerica demonstrated the ability to produce bioactive agents with pharmaceutical potential, and may provide a new lead in the pursuit of new biological sources of drug candidates.