Macromolecular crowding, the effects caused by high concentrations of macromolecules in solution, is believed to influence diffusion processes, intermolecular interactions, protein folding, and intracellular transport in living cells. The goal of this thesis was to compare translational and rotational diffusion in crowded environments to examine the effects of varying concentrations of different macromolecules on diffusion. Previous attempts have been made to characterize the effect of crowding, yet most have been unable to compare translation and rotational diffusion, and none have used model systems that offer direct comparison to other spectroscopic techniques. Using time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy, we have monitored changes to the diffusion of multiple fluorescent tracers in the presences of synthetic macromolecules and proteins. These results provide new insights into the effects of crowding on multiscale diffusion, nonspecific binding, and the local heterogeneity of microviscosity experienced by different fluorophores that allow for a meaningful comparison to other spectroscopic techniques.