Experimental charge density analysis combined with the quantum crystallographic technique of X-ray wavefunction refinement (XWR) provides quantitative insights into the intra- and intermolecular interactions formed by acetazolamide, a diuretic drug. Firstly, the analysis of charge density topology at the intermolecular level shows the presence of exceptionally strong interaction motifs such as a DDAA–AADD (D-donor, A-acceptor) type quadruple hydrogen bond motif and a sulfonamide dimer synthon. The nature and strength of intra-molecular S⋯O chalcogen bonding have been characterized using descriptors from the multipole model (MM) and XWR. Although pure geometrical criteria suggest the possibility of two intra-molecular S⋯O chalcogen bonded ring motifs, only one of them satisfies the “orbital geometry” so as to exhibit an interaction in terms of an electron density bond path and a bond critical point. The presence of ‘σ-holes’ on the sulfur atom leading to the S⋯O chalcogen bond has been visualized on the electrostatic potential surface and Laplacian isosurfaces close to the ‘reactive surface’. The electron localizability indicator (ELI) and Roby bond orders derived from the ‘experimental wave function’ provide insights into the nature of S⋯O chalcogen bonding.