A technique using horizontal attenuated total reflection (HATR) by Fourier transform infrared (FTIR) spectrometry was demonstrated for the determination of effective diffusion coefficient (De) of an inert solute in biofilm. Glucose was the selected solute, and agarose (1%) hydrogel films containing various concentrations of activated sludge biomass were used to simulate the biofilm. The agarose films were formed on the surface of an internal reflection crystal in contact with a bulk solution containing 0.5M glucose. Glucose molecules diffused through the film by concentration gradient, and the glucose concentration at the biofilm-crystal interface was measured over time by HATR-FTIR. Based on the glucose concentration and film thickness, the De of glucose was calculated according to the Fick's Law. Results showed that De of glucose in the biomass-free agarose films averaged 6.46 +/- 0.21 x 10(6) cm(2) s(-1), which is about 5% lower than the average reported De of glucose in water. The De of glucose decreased with increasing biomass concentration in the artificial biofilm. For the agarose films containing 0.45%, 0.90% and 1.80% of biomass, the De of glucose were lowered to 6.38 +/- 0.22 x 10(-6), 6.08 +/- 0.23 x 10(-6) and 5.62 +/- 0.17 x 10(4) cm(2) s(-1), respectively.