Duckweed (Lemna minor L.) is a potential biofilter for nutrient removal and acts as a substrate for heterotrophic bacteria in recirculating aquaculture systems (RAS). Here, we determined the effects of harvesting frequency of duckweed on heterotrophic bacteria in RAS. Twelve independent RAS consisting of fish-rearing tank, biofilter tank and waste-collection tank were used to study the interactions between duckweed harvest frequencies up to 6days and the composition, abundance and diversity of heterotrophic bacteria. After 36days, heterotrophic bacteria in the biofilter tank were primarily Gram-negative cocci or ovoid, coccobacilli, Gram-negative bacilli and Gram-positive bacilli. Most bacterial genera were Bacillus and Pseudomonas while the least common was Acinetobacter. Duckweed harvested after every 2days produced the highest specific growth rates (SGR) and total harvested biomass of duckweed, but harboured less abundant bacteria, whereas 6-day harvests had a higher growth index (GI) of duckweed than 2-day harvests, but caused a poor relationship between SGR and biomass harvest with the abundance and diversity of heterotrophic bacteria. Stronger correlations (R(2) >0·65) between duckweed SGR and biomass harvest with the heterotrophic bacteria diversity were observed at 4-day harvest frequency and the control. This study provides significant information on the interaction between the harvest frequency of duckweed and the composition, abundance and diversity of heterotrophic bacteria in recirculating aquaculture systems (RAS). Different harvest frequencies significantly influence the abundance and diversity of heterotrophic bacteria, which in turn may influence the nitrogen uptake efficiency of the system. The research is useful in improving the efficiency of removing nitrogenous metabolites in RAS directly by the duckweed and associated heterotrophic bacteria.