Natamycin (NATA) is a widely used food preservative. It is a non-fluorescent compound; hence, a derivatization reaction to produce a fluorescent derivative was necessary for its analysis. In this context, two derivatization reactions were applied to develop two spectrofluorimetric methods for determining NATA. Method I relies on Hantzsch condensation reaction between the primary amine group of NATA and acetylacetone-formaldehyde (AAF) reagent to produce a highly fluorescent dihydropyridine derivative that is measured at Îťem/Îťex of 481/415 nm, while Method II based on the reaction of the primary amine group of NATA with o-phthalaldehyde in the presence of N-acetylcysteine which resulted in isoindole fluorophore derivative that can be monitored at Îťem/Îťex of 433/338 nm. The experimental variables of both methods were optimized using full-factorial designs. Under the optimum conditions, the proposed methods enabled the determination of NATA over concentration ranges of 50.0â500.0 ng mLâ1 (LOD = 13 ng mLâ1) for Method I and 0.50â10.0 Îźg mLâ1 (LOD = 0.16 Îźg mLâ1) for Method II. Both methods were efficiently applied to assay NATA in ophthalmic eye drops. The application was extended to include bread and environmental water samples based on the nano-scale sensitivity of Method I.