Used in microbiology as a selective agent in culture media to identify bacteria that can hydrolyze esculin, particularly in the bile esculin agar test. Organisms like Enterococcus, group D Streptococcus, and some Listeria species break down esculin in the presence of bile and ferric ions, producing a dark precipitate that confirms their identity.

Also serves as a fluorescent marker in biochemical assays due to its natural fluorescence under UV light. Employed in research settings to study enzyme activity, especially β-glucosidase, and in the development of analytical methods for detecting glycosidase enzymes.

In cosmetics, esculin hydrate is utilized for skin lightening and reducing hyperpigmentation by inhibiting tyrosinase, an enzyme involved in melanin synthesis. It also exhibits antioxidant properties, helping to protect skin from free radical damage.

Emerging pharmaceutical research explores its anti-inflammatory and neuroprotective effects, potentially for developing health care products.