The reaction between 5-nitroisatin and phenylhydrazine in acidic ethanol yields the title compound, C14H10N4O3, whose molecular structure deviates slightly from a planar geometry (r.m.s. deviation = 0.065 Å for the mean plane through all non-H atoms). An intramolecular N—H.O hydrogen bond is present, forming a ring of graph-set motif S(6). In the crystal, molecules are linked by N—H.O and C—H.O hydrogen-bonding interactions into a two-dimensional network along (120), and rings of graph-set motif R22(8), R22(26) and R44(32) are observed. Additionally, a Hirshfeld surface analysis suggests that the molecules are stacked along [100] through C=O.Cg interactions and indicates that the most important contributions for the crystal structure are O.H (28.5%) and H.H (26.7%) interactions. An in silico evaluation of the title compound with the DHFR enzyme (dihydrofolate reductase) was performed. The isatin–hydrazone derivative and the active site of the selected enzyme show N—H.O(ASP29), N—H.O(ILE96) and Cg.Cg(PHE33) interactions.