Cryptococcal infection commonly begins as an opportunistic infection in humans, however, this can escalate to a systemic or life-threatening form in immunocompromised individuals. Here, we aim to identify novel antifungal molecules from plants resources. Sclareolide, a phytochemical classified as a sesquiterpene lactone, was assessed against Cryptococcus neoformans H99. Sclareolide exhibited promising antifungal properties with a minimum inhibitory concentration (MIC) of 16 mu g/mL. Additionally, the C. neoformans growth rate was significantly affected by sclareolide treatment in a concentration-dependent manner, as observed through a time killing assay, with a significant reduction at MIC x 8 compared to the control by 48 h. To elucidate the underlying mechanisms of sclareolide antifungal activity, fluorescence-based methods were employed. Propidium iodide (PI) accumulation assay indicated a reduction in C. neoformans membrane integrity, with values as low as 6.62 +/- 0.18% after treatment. Moreover, sclareolide at MIC x 4 and MIC x 8 significantly increased the production of reactive oxygen species (ROS) and reduced the mitochondrial membrane potential (MMP), suggesting oxidative stress and mitochondrial dysfunction in C. neoformans. Sclareolide did not induce caspase-dependent apoptosis, suggesting a non-apoptotic mechanism. Further, a checkerboard experiment was performed to assess potential synergistic interaction with Amphotericin B, however, no synergism was observed. Moving on, sclareolide at 128 mu g/mL did not exhibit toxicity in Galleria mellonella, further supporting its potential as a safe antifungal agent. These findings suggest that the antifungal activity of sclareolide against C. neoformans is mediated by oxidative stress. Further in vivo and pharmacokinetic studies are recommended to explore the potential of sclareolide as a prototype for the development of novel anti-cryptococcal therapies. C1 [Ganeshkumar, Arumugam; Lima, Patricia Michelle Nagai de; Junqueira, Juliana Campos] Sao Paulo State Univ Unesp, Inst Sci & Technol, Dept Biosci & Oral Diag, BR-12245000 Sao Jose Dos Campos, SP, Brazil. [Ganeshkumar, Arumugam] Saveetha Inst Med & Tech Sci SIMTS, Saveetha Sch Engn, Dept Mat Phys, Chennai 602105, Tamil Nadu, India. [Haribabu, Jebiti] Univ Atacama, Fac Med, Los Carreras 1579, Copiapo 1532502, Chile. [Haribabu, Jebiti] Chennai Inst Technol CIT, Chennai 600069, Tamil Nadu, India. [Borges, Bruno Montanari; Preite, Nycolas Willian; Loures, Flavio Vieira] Fed Univ Sao Paulo UNIFESP, Inst Sci & Technol, BR-12231280 Sao Jose Dos Campos, SP, Brazil. [Arulraj, Arunachalam] Univ Tecnol Metropolitana UTEM, Fac Ingn, Dept Electr, Ave Jose Pedro Alessandri 1242, Santiago, Chile. C3 Universidade Estadual Paulista; Saveetha Institute of Medical & Technical Science; Saveetha School of Engineering; Universidad de Atacama; Universidade Federal de Sao Paulo (UNIFESP); Universidad Tecnologica Metropolitana