Chagas disease is a serious health problem in Latin America. Hidroxymethylnitrofurazone (NFOH) is a nitrofurazone prodrug more active than nitrofurazone against Trypanosoma cruzi. However, NFOH presents low aqueous solubility, high photodecomposition and high toxicity. The present work is focused on the characterization of an inclusion complex of NFOH in 2-hydroxypropyl-β-cyclodextrin (HP-β-CD). The complexation with HP-β-CD was investigated using reversed-phase liquid chromatography, solubility isotherms and nuclear magnetic resonance. The retention behavior was analyzed on a reversed-phase C18 column, using acetonitrile–water (20/80, v/v) as the mobile phase, in which HP-β-CD was incorporated as a mobile phase additive. The decrease in the retention times with increasing concentrations of HP-β-CD enables the determination of the apparent stability constant of the complex (K = 6.2 ± 0.3 M−1) by HPLC. The solubility isotherm was studied and the value for the apparent stability constant (K = 7.9 ± 0.2 M−1) was calculated. The application of continuous variation method indicated the presence of a complex with 1:1 NFOH:HP-β-CD stoichiometry. The photostability study showed that the formation of an inclusion complex had a destabilizing effect on the photodecomposition of NFOH when compared to that of the “free” molecule in solution. The mobility investigation (by NMR longitudinal relaxation time) gives information about the complexation of NFOH with HP-β-CD. In preliminary toxicity studies, cell viability tests revealed that inclusion complexes were able to decrease the toxic effect (p < 0.01) caused by NFOH.