All molecular crystals contain defects, yet the effects these defects have on the material properties they exhibit are poorly understood. Here, the relationship between mechanical properties and defect concentration is established through nanoindentation studies on single crystals of uric acid (UA) with two different types of substitutional defects. Defects are intentionally created by preparing UA-dye crystals with two different dyes chosen to span either one or more than one molecular layer when included in the host matrix. The included dye concentrations in materials prepared from several well-defined growth conditions are then spectroscopically quantified. Sector-specific nanoindentation measurements on UA-dye crystals with dye concentrations ranging from 0.01 to 1 wt % illustrate two competing effects. UA-dye crystals with the lowest dye concentrations exhibited Young’s moduli reductions of up to ∼50% compared to undoped crystals, with defects causing elastic softening. With progressively higher concentrations of defects spanning multiple UA layers, a second competing material stiffening effect is activated. At the concentrations necessary for material strengthening to occur, twinning and other morphological changes are also observed, suggesting that the macroscopic changes are likely related to efforts to reduce lattice strain. The magnitude of the property changes realized illustrates the potential of defect engineering to tune mechanical properties and may be especially beneficial in systems where increased plasticity is desired.