The valence ionization of uracil and mixed water–uracil clusters has been studied experimentally and by ab initio calculations. In both measurements, the spectrum onset shows a red shift with respect to the uracil molecule, with the mixed cluster characterized by peculiar features unexplained by the sum of independent contributions of the water or uracil aggregation. To interpret and assign all the contributions, we performed a series of multi-level calculations, starting from an exploration of several cluster structures using automated conformer-search algorithms based on a tight-binding approach. Ionization energies have been assessed on smaller clusters via a comparison between accurate wavefunction-based approaches and cost-effective DFT-based simulations, the latter of which were applied to clusters up to 12 uracil and 36 water molecules. The results confirm that ( i) the bottom-up approach based on a multilevel method [Mattioli et al. Phys. Chem. Chem. Phys. 23, 1859 (2021)] to the structure of neutral clusters of unknown experimental composition converges to precise structure–property relationships and ( ii) the coexistence of pure and mixed clusters in the water–uracil samples. A natural bond orbital (NBO) analysis performed on a subset of clusters highlighted the special role of H-bonds in the formation of the aggregates. The NBO analysis yields second-order perturbative energy between the H-bond donor and acceptor orbitals correlated with the calculated ionization energies. This sheds light on the role of the oxygen lone-pairs of the uracil CO group in the formation of strong H-bonds, with a stronger directionality in mixed clusters, giving a quantitative explanation for the formation of core–shell structures.