The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several enzymes and is often understood in terms of changes to enzyme conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3',5,5'-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25-30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease enzyme activity and that these effects vary with the identity of the molecules involved. Because many enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic reactions in complex media such as biological fluids.