Background: Since common drug therapies cannot eradicate Candida biofilm, extensive studies are required to develop more effective antifungal compounds and identify their mechanism of action against Candida biofilm. Peganum harmala L. is a traditional medicinal plant, the seeds of which have been used to treat various diseases. Objectives: This study aimed to investigate the anti-biofilm mechanisms of P. harmala extract (PHE) and the expression of CAT1, EFG1, and BCR1 genes involved in oxidative stress response and biofilm formation in Candida albicans. Methods: Anti-biofilm activity of PHE was evaluated by crystal violet assay to determine biofilm formation on 33 C. albicans isolates. Finally, a real-time polymerase chain reaction was performed to analyze the effect of PHE on the expression of CAT1, EFG1, and BCR1 genes in C. albicans. Results: This study determined the minimum biofilm eradication concentration (MBEC) of 15 isolates in concentrations between 0.49 - 3.9 μg/mL of P. harmala extract. Statistical analysis showed that the exposure of C. albicans biofilm to PHE significantly reduced the expression of CAT1 mRNA in C. albicans isolates (P = 0.0068). However, no significant difference was observed in the expression of EFG1 and BCR1 genes. Conclusions: The results demonstrated that PHE significantly decreased CAT1 expression in C. albicans cells treated with the herbal extract. PHE is likely to accumulate hydrogen peroxide (H2O2) by reducing CAT1 expression and disrupting the pro-oxidant/antioxidant balance that leads to the overproduction of reactive oxygen species (ROS) and can cause damage to cellular components and eventually destroy C. albicans biofilm.