Nonspecific binding of crowder proteins with functional proteins is likely prevalent in vivo, yet direct quantitative evidence, let alone residue-specific information, is scarce. Here we present nuclear magnetic resonance (NMR) characterization showing that bovine serum albumin weakly but preferentially interacts with the histidine carrier protein (HPr). Notably, the binding interface overlaps with that for HPr's specific partner protein, EIN, leading to competition. The crowder protein thus decreases the EIN-HPr binding affinity and accelerates the dissociation of the native complex. In contrast, Ficoll-70 stabilizes the native complex and slows its dissociation, as one would expect from excluded-volume and microviscosity effects. Our atomistic modeling of macromolecular crowding rationalizes the experimental data and provides quantitative insights into the energetics of protein-crowder interactions. The integrated NMR and modeling study yields benchmarks for the effects of crowded cellular environments on protein-protein specific interactions, with implications for evolution regarding how nonspecific binding can be minimized or exploited.