Candida spp. is one of the most common opportunistic human fungal pathogens, responsible for 90–100% of mucosal infections. Germ tube formation, hyphal morphogenesis, the production of tissue-damaging extracellular enzymes, and drug-resistant biofilm formation contribute to their pathogenicity, which can lead to systemic infections in the worst scenarios. Thus, there is an urgent need to discover new therapeutic agents to overcome the above virulence factors. Therefore, we aimed to prepare Crinum latifolium leaves-mediated biosynthesis of gold nanoparticles (AuNPs) that was characterized by various sophisticated techniques, and further their antifungal, antibiofilm, and anti-virulence activities was investigated. The AuNPs show a zone of inhibition between 19 and 22 mm for test strains of Candida spp. at 1000 µg/ml, whereas the MIC values were ranged from 250 to 500 µg/ml. AuNPs inhibit germ tube formation in C. albicans by 93.3% at 50 µg/ml. Furthermore, exposure to AuNPs significantly reduced the secretions of phospholipase, proteinase, hemolysin, esterase, and lipase. Confocal laser scanning microscopy (CLSM) investigation shows that 25 µg/ml of AuNPs significantly inhibit colonization and biofilm formation. Lastly, the interaction of Candidal cells with AuNPs revealed, ultrastructural changes in the cell wall and cell membranes as visualized by a transmission electron microscope (TEM).