Molecular self-assembly of the minimal diphenylalanine (Phe-Phe) peptide building block shows unique morphological organizations and potential utility in biochemistry and biomaterials applications. Furthermore, the molecular engineering of nucleoside-conjugated Phe-Phe scaffolds allows the formation of diverse architectures with tunable biophysical properties. While the self-assembly of homochiral l-dipeptides is well characterized, stereochemistry is known to determine the conformation, which also governs self-assembly through molecular packing effects. Here, the effect of stereochemistry and hydrophobicity on Phe-Phe nucleoside conjugates using all four diastereomers [(l)Phe-(l)Phe, (d)Phe-(dPhe, (l)Phe-(d)Phe, and (d)Phe-(l)Phe] of Phe-Phe conjugates is systematically studied. The homochiral peptides form well-defined nanorods while the heterochiral dipeptides do not form any regular structures. Since heterocyclic nucleobases can self-assemble through hydrogen-bonded complementary base-pairing, the self-assembly of chiral nucleoside-conjugated Phe-Phe peptides is examined. All conjugated Phe-Phe peptides form seamless spherical particles. The completely or partially deprotected peptides do not assemble to any defined nanostructures suggesting that self-assembly is governed by the precise hydrophobic/hydrophilic balance in the assembling units. Contact angle measurements of the diastereomeric peptides reveal a subtle difference in stereochemistry-dependent molecular hydrophobicity. Taken together, it is revealed that the combination of chirality together with hydrophobic/hydrophilic balance within the peptides dictates the self-assembly of Phe-Phe dipeptide nucleoside conjugates.