The effects of increased crowding and confinement on the mobility of individual fluorescent molecules were studied using Fluorescence Correlation Spectroscopy (FCS) in a microfluidic device with sealable femtolitre-volume chambers, and compared to three dimensional stochastic Monte Carlo simulations. When crowding and the degree of confinement were increased simultaneously, extended correlation times of fluorescent intensity fluctuations were observed with FCS compared to varying either crowding or confinement alone. Both experimental data and simulation suggest these extended correlation times were due to increased fluorophore adsorption–desorption events at the chamber lid in the presence of crowders. The data in increasingly confined and crowded chambers described here captures some of the salient features of crowding in cell-like environments.