Living cells are crowded with macromolecules and organelles. Yet, it is not fully understood how macromolecular crowding affects the myriad of biochemical reactions, transport and the structural stability of biomolecules that are essential to cellular function and survival. These molecular processes, with or without electrostatic interactions, in living cells are therefore expected to be distinct from those carried out in test tube in dilute solutions where excluded volumes are absent. Thus there is an urgent need to understand the macromolecular crowding effects on cellular and molecular biophysics towards quantitative cell biology. In this report, we investigated how biomimetic crowding affects both the rotational and translation diffusion of a small probe (rhodamine green, RhG). For biomimetic crowding agents, we used Ficoll-70 (synthetic polymer), bovine serum albumin and ovalbumin (proteins) at various concentrations in a buffer at room temperature. As a control, we carried out similar measurements on glycerolenriched buffer as an environment with homogeneous viscosity as a function of glycerol concentration. The corresponding bulk viscosity was measured independently to test the validity of the Stokes-Einstein model of a diffusing species undergoing a random walk. For rotational diffusion (ps-ns time scale), we used time-resolved anisotropy measurements to examine potential binding of RhG as a function of the crowding agents (surface structure and size). For translational diffusion (μs-s time scale), we used fluorescence correlation spectroscopy for single-molecule fluctuation analysis. Our results allow us to examine the diffusion model of a molecular probe in crowded environments as a function of concentration, length scale, homogeneous versus heterogeneous viscosity, size and surface structures. These biomimetic crowding studies, using non-invasive fluorescence spectroscopy methods, represent an important step towards understanding cellular biophysics and quantitative cell biology.