Free nitrous acid (FNA) is a promising chemical reagent for excess sludge reduction. The distinctive properties of FNA treatment on waste activated sludge (WAS) disposal have previously been demonstrated, however, the cellular response, permeabilization, and disruption caused by low-concentration FNA and the direct cell solubilization of WAS using concentrated FNA should be better understood. In this study, the parameters that influence the sludge solubilization efficiency were optimized over a wide range of FNA concentrations. The sludge solubilization efficiency was found to be superior when the sludge was exposed to FNA (when the dosage of NaNO₂ was 0.12 g g⁻¹ TSS and the pH was 3.0, FNA = 20.94 mg L⁻¹) for 10 h at 25 °C, and the TSS removal and COD dissolution efficiencies were found to be prominent at 38% and 7%, respectively. In the FNA treatment of WAS, some FNA-tolerable cells increased the K⁺, Ca²⁺, and H⁺ effluxes under low concentrations of FNA, and finally achieved ion homeostasis based on the results using a scanning ion-selective electrode measurement technique. This could cause the cells in WAS to maintain cytoactivity and integrity under a low-concentration FNA treatment. Furthermore, flow cytometry was used to assess the permeabilization and disruption of sludge cells toward a concentration gradient of FNA. Flow cytometry results indicated that cells in sludge flocs were disrupted within 30 minutes when the FNA concentration was above 8 mg L⁻¹.