<p>Nitrification plays a crucial role in global nitrogen cycling and treatment processes. However, the relationships between the nitrifier guilds of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are still poorly understood, especially in freshwater habitats. This study examined the physiological interactions between the AOB and NOB present in a freshwater aquarium biofilter by culturing them, either together or separately, in a synthetic medium. Metagenomic and 16S rRNA gene sequencing revealed the presence and the draft genomes of Nitrosomonas-like AOB as well as Nitrobacter-like NOB in the cultures, including the first draft genome of Nitrobacter vulgaris. The nitrifiers exhibited different growth rates with different ammonium (NH<sub>4</sub><sup>+</sup>) or nitrite concentrations (50–1,500 μM) and the growth rates were elevated under a high bicarbonate (HCO<sub>3</sub><sup>-</sup>) concentration. The half-saturation constant (K<sub>s</sub> for NH<sub>4</sub><sup>+</sup>), the maximum growth rate (μ<sub>max</sub>), and the lag duration indicated a strong dependence on the synergistic relationships between the two guilds. Overall, the ecophysiological and metagenomic results in this study provided insights into the phylogeny of the key nitrifying players in a freshwater biofilter and showed that interactions between the two nitrifying guilds in a microbial community enhanced nitrification.</p>