Allylthiourea (ATU) and chlorate (ClO3-) are often used to selectively inhibit nitritation and nitratation. In this work we identified challenges with use of these compounds in inhibitory assays with filter material from a biological rapid sand filter for groundwater treatment. Inhibition was investigated in continuous-flow lab-scale columns, packed with filter material from a full-scale filter and supplied with NH4+ or NO2-. ATU concentrations of 0.1-0.5 mM interfered with the indophenol blue method for NH4+ quantification leading to underestimation of the measured NH4+ concentration. Interference was stronger at higher ATU levels and resulted in no NH4+ detection at 0.5 mM ATU. ClO3- at typical concentrations for inhibition assays (1-10 mM) inhibited nitratation by less than 6%, while nitritation was instead inhibited by 91% when NH4+ was supplied. On the other hand, nitratation was inhibited by 67-71% at 10-20 mM ClO3- when NO2- was supplied, suggesting significant nitratation inhibition at higher NO2- concentrations. No chlorite (ClO2-) was detected in the effluent, and thus we could not confirm that nitritation inhibition was caused by ClO3- reduction to ClO2-. In conclusion, ATU and ClO3- should be used with caution in inhibition assays, because analytical interference and poor selectivity for the targeted process may affect the experimental outcome and compromise result interpretation.