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The rapid emergence of invasive infections caused by azole-resistant Candida tropicalis has become a public health concern, and there is an urgent need for alternative treatment strategies. Studies have demonstrated the antibacterial effects of nisin, a well-known peptide naturally produced by Lactococcus lactis subsp. lactis. However, there is scant information about the antifungal effect of nisin against C. tropicalis. The present study aims to investigate the in vitro antifungal activity of nisin against clinical isolates of azole-resistant C. tropicalis strains, as well as its inhibitory effect on biofilm formation. A total of 35 C. tropicalis strains isolated from patients with invasive fungal infections were divided into the azole-resistant group and the azole-sensitive group, containing 21 and 14 strains, respectively. The relative expression levels of the ERG11 and UPC2 genes in the azole-resistant group were higher than those in the azole-sensitive group (p