The present study helps us to understand the effects of temperature and viscosity on bioprocesses in immobilized cell bioreactors. Glucose diffusivities through Saccharomyces cerevisiae biofilms are experimentally investigated at variable biofilm thickness, temperature, and concentration of carboxymethylcellulose (CMC) used as viscosity-raising additive. Comparing these experimental values with those for free diffusion of glucose in water shows us that biofilm strongly reduces diffusivity (about 74%), mainly due to a decrease in the effective diffusion area. Only a slight increase in diffusivity is observed when biofilm thickness is increased. In addition, the results obtained in this work demonstrate that the well-known empirical equations of Wilke and Chang, and of Guzman and Andrade can be combined to a relationship which takes into account the simultaneous efforts of temperature and viscosity on the effective glucose diffusivity. This equation appears to be valid in glucose aqueous solutions whether or not biofilm is used, whereas it is not applicable when viscosity-raising additives, such as CMC, are used.