π-π Stacking is crucial for stabilizing molecular structures and facilitating interactions in biological systems and chemical processes. The presence of electron-withdrawing and electron-donating substituents, particularly the hydroxyl group, significantly influences the energetics and energetically favorable configurations of π-stacked complexes. In this study, we utilized UV–Vis absorption and resonance Raman spectroscopies, alongside density functional theory (DFT) calculations, to investigate the competition between dissociation and self-aggregation of 4-nitro-1-naphthol (NNH) in various polar solvents. Our results demonstrate the self-aggregation of NNH in polar solvents and identify the most likely dimer configuration, which is antiparallel π-stacked and stabilized by hydrogen bonds between hydroxyl and nitro groups. Given its broad applications in mass spectrometry, environmental remediation, and aerosol tracking, a comprehensive understanding of the molecular interactions and structural properties of NNH is essential for its effective use in various scientific fields.