Background <p><i>Candida albicans</i> (<i>C. albicans</i>) is a major opportunistic fungal pathogen responsible for both invasive and superficial infections, including vulvovaginal candidiasis (VVC), which affects a large proportion of women worldwide and is prone to recurrence. Limitations of conventional antifungal therapies and the emergence of drug resistance highlight the urgent need for novel therapeutic agents. D-lactic acid (D-LA), a chiral organic acid naturally produced by <i>Lactobacillus</i> spp., has been proposed as a potential antifungal candidate. However, its activity against <i>C. albicans</i> and underlying mechanisms remain unclear. Therefore, this study aimed to evaluate the antifungal activity of D-LA against <i>C. albicans</i> and to explore its potential mechanisms.</p> Methods <p><i>C. albicans</i> SC5314 was treated with D-LA in vitro to evaluate its effects on fungal growth, hyphal morphogenesis, and biofilm development using broth microdilution assays, hyphal induction/inhibition assays, crystal violet staining, and XTT-based metabolic activity measurement. Cell membrane integrity was assessed via trypan blue exclusion and live/dead fluorescent staining. RNA-seq and qRT-PCR were performed to identify differentially expressed genes and enriched functional pathways. The antifungal efficacy of D-LA was further evaluated in a mouse model of vaginal candidiasis.</p> Results <p>D-LA significantly inhibited hyphal formation and biofilm development in a concentration-dependent manner. Crystal violet and XTT assays confirmed substantial suppression of biofilm biomass and metabolic activity. Live/dead staining and trypan blue exclusion indicated increased cell membrane damage after D-LA exposure. Transcriptomic profiling revealed significant changes in genes related to hyphal development, cell wall organization, iron metabolism, acid phosphatase activity, and stress response. qRT-PCR validation demonstrated that the addition of D-LA downregulated virulence-associated genes (<i>ECE1</i>,<i> TEC1</i>,<i> ALS1</i>) and upregulated the pH-regulated gene <i>PHR2</i> in <i>C. albicans</i>. Cytotoxicity testing on VK2/E6E7 vaginal epithelial cells indicated minimal toxicity at concentrations effective against <i>C. albicans</i>. In vivo antifungal efficacy and safety of D-LA in a mouse model of <i>C. albicans</i> vaginal infection demonstrating potent efficacy and a favorable profile.</p> Conclusion <p>D-LA exhibits potent antifungal activity against <i>C. albicans</i> by disrupting hyphal formation, biofilm development, and cell wall integrity, accompanied by transcriptional reprogramming of virulence-associated pathways. Its low cytotoxicity towards vaginal epithelial cells and mouse vaginal mucosa supports its potential as a safe alternative or adjunctive agent for VVC management.</p>