Favorable molecular interactions between group 16 elements have been implicated in catalysis, biological processes, and materials and medicinal chemistry. Such interactions have since become known as chalcogen bonds by analogy to hydrogen and halogen bonds. Although the prevalence and applications of chalcogen-bonding interactions continues to develop, debate still surrounds the energetic significance and physicochemical origins of this class of sigma-hole interaction. Here, synthetic molecular balances were used to perform a quantitative experimental investigation of chalcogen-bonding interactions. Over 160 experimental conformational free energies were measured in 13 different solvents to examine the energetics of O center dot center dot S, O center dot center dot Se, S center dot center dot center dot S, O center dot center dot center dot HC, and S center dot center dot center dot HC contacts and the associated substituent and solvent effects. The strongest chalcogen-bonding interactions were found to be at least as strong as conventional H-bonds, but unlike H-bonds, surprisingly independent of the solvent. The independence of the conformational free energies on solvent polarity, polarizability, and H-bonding characteristics showed that electrostatic, solvophobic, and van der Waals dispersion forces did not account for the observed experimental trends. Instead, a quantitative relationship between the experimental conformational free energies and computed molecular orbital energies was consistent with the chalcogen-bonding interactions being dominated by n -> sigma* orbital delocalization between a lone pair (n) of a (thio)amide donor and the antibonding c orbital of an acceptor thiophene or selenophene. Interestingly, stabilization was manifested through the same acceptor molecular orbital irrespective of whether a direct chalcogen chalcogen or chalcogen center dot center dot center dot H-C contact was made. Our results underline the importance of often-overlooked orbital delocalization effects in conformational control and molecular recognition phenomena.