Maternal colonization by Group B Streptococcus during pregnancy increases the risk of neonatal infection due to vertical transmission from mother to fetus before or during labor. The aims of this study were to evaluate the antimicrobial activity of SP80 (obtained from RGE) and its synergism associated with the antibiotic against strains of Streptococcus agalactiae. Biomonitoring of SP80 disclosed antimicrobial activity only in fractions F18, F19, F20 and F42. The broth microdilution was used to determine the antimicrobial activity of SP80 and fractions from SP80 and to establish the MIC of SP80 (2.40 mg/mL). By using the disk diffusion method, fifty-five clinical isolates of S. agalactiae and 1 ATCC were tested against the association of SP80 with antibiotic penicillin G and ampicillin, respectively, for synergistic assessment. The association of SP80 with penicillin G showed that the mean of the inhibition halos decreased, but it was not significant, with p<0.07. In contrast, the association of SP80 with ampicillin caused the mean inhibition halos to increase with a p<0.001, a significant result. SP80 has antimicrobial activity against S. agalactiae Gram-positive bacteria, and the association with the antibiotic ampicillin showed a synergistic effect, which did not occur when in association with penicillin G.