This data set contains raw video files (.avi) of gold spherical nanoparticles with diameter of 100nm diffusing above a human keratinocyte cell layer (HaCaT) in standard cell culture solutions (Dulbecco’s modified Eagle medium supplemented with 10% or 0% fetal bovine serum). Nanoparticles were visualised using the technique developed by Patterson and Whelan (2008) using a Zeiss Axio Observer inverted microscope. Videos were taken using the Zeiss Axio Cam 305 mono and 40x objective lens. The pixel size is 0.086µm x 0.086µm. T1, 2 and 3 refers to three timepoints which were 15, 60, and 105 minutes respectively. H1, 2, 3 and 4 refer to height ranges above the top surface of the monolayer at which nanoparticles were tracked which were 20-25, 45-50, 70-75 and 95-100µm. Nanoparticles were visualised whilst inside an incubator stage maintained at 37C throughout the tracking process.