........ Oxygen consumption rates of biofilm-associated bacteria were measured in closed metabolic chambers under standard conditions. The protocol included sequential flushes of either pure tap water, or tap water spiked with nitrite, ammonium or acetate. The results showed consistent carrier-specific metabolic activities of endogenous respiration, nitrite-oxidizing bacteria (NOB), ammonia-oxidizing bacteria (AOB) and heterotrophic bacteria (HB) in biofilm. The highest activities normalized to volumetric oxygen consumption rates, were found in polymeric foam with values of 677 ± 125, 764 ± 156 and 166 ± 36 g O2·m−3·d−1 for NOB, AOB and HB, respectively. The biofilter performance evaluation based on respirometric data showed satisfactory accordance with the values from substrate degradation batch kinetic tests for all three tested carriers, confirming the feasibility and robustness of the method as a means to assessing biofilter performance. Our study provides an on-site tool in biofilter performance tests and leads to a better understanding of dynamic and relationship between biofiltration and water quality. ........