Paddlewheel diruthenium complexes have interesting pharmaceutical properties. The diruthenium tetracetate complex ([Ru₂Cl(O₂CCH₃)₄]), the prototype of the diruthenium compound family, has been used to prepare promising anticancer agents (i.e., against glioma tumor models and glioblastoma). The interaction of [Ru₂Cl(O₂CCH₃)₄] with proteins has been already investigated: diruthenium moieties bind Asp side chains upon releasing of one acetate ligand; a second acetate is replaced by two water molecules in each diruthenium center. Recently, it has been suggested that the use of bulky equatorial substituents may constitute an approach to increase the selectivity of diruthenium complexes toward anticancer targets. To study the effect of equatorial ligand replacement on the reactivity of diruthenium compounds with proteins, we solved high-resolution X-ray structures of adducts formed upon reaction of the model protein lysozyme with some monosubstituted complexes [Ru₂Cl(L-L)(O₂CCH₃)₃] (L-L = N,N’-donor or N,O-donor bridging ligands). Results indicate that these complexes bind the protein via coordination of the Ru-Ru core to the side chain of Asp residues at the equatorial coordination site, losing only one acetate ligand. Protein binding occurs cis or trans to the L-L ligands that remain attached to the dimetallic center. The side chain of a Lys and even main chain carbonyl groups can coordinate diruthenium core at the axial site. Data help to understand the reactivity of paddlewheel diruthenium complexes with proteins, providing useful information for the design of new diruthenium compounds with improved pharmacological properties.