The kinetic behavior of signaling pathways and other biochemical reaction networks have traditionally been characterized within the framework of the Michaelis-Menten formulism, which is derived from chemical kinetics based on the laws of mass-action appropriate for dilute, homogeneous solutions. However, evidence has indicated a breakdown of classical mass-action kinetics for reactions under dimensionally-restricted, confined or crowded conditions, such as those that occur in the in vivo nanoenvironments found inside a cell or cellular compartment. We have fabricated femtoliter-scale biomimetic compartments in microfluidics-based devices for capturing real-time single-molecule enzyme dynamics in confined and crowded spaces. We have found that fluctuation-dominated kinetic behaviors in single-enzyme reactions become increasingly important as the degree of confinement and crowding increase. This suggests that in addition to fluctuating enzyme conformation, stochastic fluctuations in local reactant concentrations in strongly diffusion-limited environments are responsible for the complex behavior seen in single-molecule enzyme kinetics