Trichophyton rubrum is the most common cause of dermatophyte skin infections in humans worldwide. The aim of the current study is to search for effective and safe antifungal agents by investigating the bioactivity of Pimpinella anisum and Moringa oleifera extracts, including the impact on the keratinase gene expression of T. rubrum. Astragalin and cryptochlorogenic acid were detected in M. oleifera leaf extracts by Gas chromatography-mass spectrometry (GC-MS), whereas anethole was extracted from P. anisum oil. The expression of the keratinase gene was assessed in two strains of T. rubrum (KP979791 and KP979787) in Sabouraud Dextrose Broth (SDB) medium (containing 10% keratin) separately supplemented with plant extracts fractions. The results showed that cryptochlorogenic acid and astragalin were active compounds (molecular weights of 353.26 and 449.35 g/mol, respectively that considerably down-regulated the keratinase genes in both strains of T. rubrum. M. oleifera crude extract, astragalin, cryptochlorogenic acid, anise oil, and anethole resulted in the down-regulation of the keratinase genes of T. rubrum strain KP979791 by 0.048, 0.291, 0.001, 0.007, and 0.009 (fold-change), respectively, and that of strain KP979787 by 0.151, 0.42, 0.16, 0.079, and 0.092 as compared to the control (1.00). Nearly all of these outcomes are novel findings that may aid in the identification of novel effective drug targets and antifungal agents.