Nystatin is a fungicidal and fungistatic polyene antifungal targeting Candida species, especially those responsible for vaginal infections. It functions by inhibiting ergosterol synthesis and increasing cell membrane permeability, ultimately leading to fungal cell death. Nystatin has been used in combination with erythromycin and streptomycin in cell culture to eliminate fungal contamination.

Nystatin is often used in clinical in vitro tests known as antimicrobial susceptibility tests or ASTs to determine their efficacy against certain bacterial species. They are tested against gram-negative and gram-positive bacteria using panels, discs, and MIC strips by medical microbiologists. ASTs decrease the risk of using an antibiotic against bacteria exhibiting resistance to it, and the results are used in clinical settings to determine which antibiotic(s) to prescribe for various infections.