<p>Partial nitrification (PN) is a highly promising strategy for energy- and carbon-efficient wastewater treatment. Effectively suppressing nitrite-oxidizing bacteria (NOB) is a critical challenge for stable PN operation. This work presents a NOB inhibition strategy on the basis of intrinsic substances in wastewater, namely salt and free ammonia (FA). Three different start-up strategies were adopted: 1) first increasing NH₄⁺-N and then increasing salinity, 2) first increasing NH₄⁺-N and then increasing salinity, and 3) increasing NH₄⁺-N and salinity simultaneously. Either FA or salinity alone caused a temporary decrease in nitrite oxidation efficiency, resulting in a nitrite accumulation rate (NAR) &lt; 35%. Under their combined effect, NAR &gt; 80% was achieved beginning on day 8 (0.61 mg FA/L and 5 g NaCl/L), with a sustained increase in NAR, demonstrating the synergistic inhibitory effect of salinity and FA on NOB activity. In contrast, the enzymatic activity of ammonia-oxidizing bacteria (AOB) increased by 159.5%–203.9% due to the increased concentration of the substrate NH₄⁺-N. In response to the three combined inhibition strategies, polysaccharide excretion was stimulated in all stratified extracellular polymeric substances, while the synergistic effect of FA and salinity further enhanced protein excretion. Following PN initiation with the three strategies, <i>Nitrospira</i>, the only NOB observed, was nearly washed out, and <i>Nitrosomonas</i> became the dominant AOB. The strategy of simultaneously increasing FA and salinity led to the greatest abundance of <i>Nitrosomonas</i> (28.42%). This study offers a novel strategy to achieve PN, which is beneficial for promoting PN-based carbon-efficient wastewater treatment.</p>