Fungal infections are a significant healthcare burden globally. With only four main classes of antifungals available and increasing antifungal resistance, it is imperative to explore novel antifungal treatments. Cryptococcus neoformans and Cryptococcus gattii are the most common causes of cryptococcosis, a life-threatening fungal infection of humans and various other animals including cats, dogs and livestock. Previously, our group has established that CBD and CBDV, two non-psychoactive phytocannabinoids, have antifungal activity against human clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex as well as the laboratory reference strain C. neoformans H99. Morphological analysis and proteomics indicated that phytocannabinoid fungicidal activity involved disruption of the cell membrane and reduction of capsule thickness, however, did not fully elucidate the mechanism of action. This project explores the breadth and utility of phytocannabinoid antifungal activity, with two distinct aims. The first is to expand the antifungal testing screen to encompass animal isolates of Cryptococcus species and assess whether phytocannabinoids can be useful in veterinary and/or agricultural settings. The second is to further define the mechanism of action of the phytocannabinoids against Cryptococcus species utilising a C. neoformans knockout mutant library to identify molecular targets, as well as further pinpointing targets of membrane disruption using fluorescent dyes. These findings will enable progression of the development of phytocannabinoids as novel antifungal therapies for both human and veterinary cryptococcal infections.