Thevetia peruviana is an ornamental plant considered source of biologically compounds with cardiac and antimicrobial activity. These compounds are normally extracted from different parts of the fully growth plants. In this work, extracts were obtained from callus and cell suspension cultures of T. peruviana and their antimicrobial activity was evaluated by disk diffusion tests against gram negative (Salmonella thipimurium and Escherichia coli) and gram positive (Staphylococcus aureus and Bacillus cereus) strains. Ethanol, methanol and hexane extracts from callus and cell suspension cultures showed biological activity. Methanolic cell suspension extract showed activity against B. cereus and S. aureus. Ethanolic cell suspension extract inhibit all the bacteria, especially S. thipimurium while hexanic extract showed resistance activity against S. thipimurium, S. aureus and B. cereus. In terms of the source of the extracts, hexane extracts obtained from cell suspension cultures showed a higher antimicrobial activity compared to callus, while ethanol extracts had an inverse behavior. These results outline in vitro cell culture of T. peruviana as a feasible biotechnological platform for the production of compounds with antimicrobial activity.