Background: The present work aimed to investigate the effect of Peganum harmala L. in increasing the growth and production of secondary metabolites of Streptomyces ayarius strain S115 via ISP2 culture medium. Peganum harmala L. was dried and added to ISP2 medium. The morphological properties and the antibacterial activity of S. ayarius strain S115 seeded in ISP2 and the modified ISP2 media was eval-uated by using the agar well diffusion method against five pathogenic bacteria, Escherichia coli Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia sp. and Methicillin-Resistant Staphylococcus aureus. The biomass of S. ayarius strain S115 in both media was determined. The kinetics of growth and production of secondary metabolites were studied for 7 d in different carbon sources. Results: Culture on the modified ISP2 showed an effective growth of S. ayarius strain S115 with changed color of the aerial mycelium from gray to white. The antibacterial activity revealed large inhibition zones against the tested pathogenic bacteria compared to those of the ISP2. The amount of S. ayarius strain S115 biomass was twice as high in the modified ISP2. The effect of different carbon sources on the growth and production of secondary metabolites of S. ayarius strain S115 revealed the highest biomass and biological activities through using glucose in the modified ISP2 on the 3rd day of culture. Of note, glycerol was found as the optimal carbon source in ISP2.Conclusions: The discovery of new bioactive molecules from actinomycetes seeded in specific culture media may contribute to the development of therapeutic solutions How to cite: Nait Marzoug A, Ayari A, Khaldi F, et al. Effect of Peganum harmala L. extract supplemented ISP2 medium on growth and production of secondary metabolites of Streptomyces ayarius S115. Electron J Biotechnol 2023;64. https://doi.org/10.1016/j.ejbt.2022.12.006. & COPY; 2023 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).