The current study was designed to evaluate bioactive compounds under the influence of biotic stress on seedlings of Peganum harmala. Biologically active compounds were determined by using different techniques. The HPLC and GC-MS analyses detected the significant amount of phenolic acids and active biological compound. Total protein content, activity of proteases, α-amylases, catalases and peroxidases were observed to be accelerated under fungal stress. The seedling extracts exhibited prominent antifungal and antimicrobial activity against selected strains. The present study showed that P. harmala is a good candidate to be used in natural therapies and medicine.