The diffusive dynamics of 100 nm to 400 nm diameter polystyrene nanoparticles dispersed in water were studied using brightfield and fluorescence based differential dynamic microscopy (DDM) and compared to those obtained from dynamic light scattering. The relaxation times measured with brightfield and fluorescence DDM over a broad range of concentration of nanoparticles (10−6 ≤ φ ≤ 10−3) and scattering vectors (0.5 μm−1 < q < 10 μm−1) are in excellent agreement with each other and extrapolate quantitatively to those obtained from DLS measurements. The diffusion coefficients extracted from the q-dependent relaxation times using all three methods are independent of the nanoparticle concentration.