The present study uses density functional theory to investigate the effects of hydrogen bonding on the acidity of C5- and C6-substituted uracil derivatives. The proton affinities and acidities of uracil donor and acceptor sites generally decrease and increase, respectively, with an increase in the electronegativity of the uracil substituent. Despite these substituent effects, the binding strengths of small molecules (NH3, H2O, or HF) to the uracil derivatives are relatively independent of the substituent, which indicates that the changes in the uracil proton affinity and acidity effectively cancel. The acidities of substituted uracil complexes increase not only with the electronegativity of the substituent, but also with the acidity of the small molecule bound to the uracil ring. However, the magnitude of the effect of hydrogen bonding on the acidity of uracil derivatives is not dependent on the nature or position of the substituent. Our results lead to a greater fundamental understanding of the effects of substituents on the hydrogen-bonding properties of uracil, which may have implications for understanding biological applications and processes that involve these modified nucleobases.