Experimental tests were carried out in order to ascertain the biomass activity in a biological fluidized bed reactor. A semi-pilot fluidized bed reactor was used with two different supports (sand and glass particles). The specific biomass activity was followed during the tests. It reached a maximum and after declined vs. biomass hold up and bed expansion. The results clearly put in evidence as the activity is strongly effected by liquid-solid mass transfer and support roughness which in turn determines the compactness of biofilm and its diffusive properties. By controlling the bed expansion it is possible to decide the macro reaction regime of work: external mass transfer, biokinetics or diffusive control according to incoming substrate concentration, in order to maximize the bioreactor performance.