AbstractFungal pathogens pose a major threat toCannabis sativaproduction, requiring safe and effective management procedures to control disease. Chitin and chitosan are natural molecules that elicit plant defense responses. Investigation of their effects onC. sativawill advance understanding of plant responses towards elicitors and provide a potential pathway to enhance plant resistance against diseases. Plants were grown in the in vitro Root‐TRAPR system and treated with colloidal chitin and chitosan. Plant morphology was monitored, then plant tissues and exudates were collected for enzymatic activity assays, phytohormone quantification, qPCR analysis and proteomics profiling. Chitosan treatments showed increased total chitinase activity and expression of pathogenesis‐related (PR) genes by 3–5 times in the root tissues. In the exudates, total peroxidase and chitinase activities and levels of defense proteins such as PR protein 1 and endochitinase 2 were increased. Shoot development was unaffected, but root development was inhibited after chitosan exposure. In contrast, chitin treatments had no significant impact on any defense parameters, including enzymatic activities, hormone quantities, gene expression levels and root secreted proteins. These results indicate that colloidal chitosan, significantly enhancing defense responses inC. sativaroot system, could be used as a potential elicitor, particularly in hydroponic scenarios to manage crop diseases.