Biofouling caused by the deposition or growth of microorganisms on the membrane surface is one of the major concerns in nanofiltration (NF) and reverse osmosis (RO) processes. Assimilable organic carbon (AOC) has been a useful index to assess the growth potential of bacteria. In the case of drinking water, the AOC assay method has been widely applied to estimate growth or regrowth potential of bacteria in distribution and storage systems. However, studies on AOC measurement for high salinity water samples such as brackish water and seawater are rather scarce. The objective of this research is to investigate the influence of water salinity on the conventional AOC assay method. AOC samples with different salt concentrations were prepared by varying NaCl concentration from 0 to 35,000 mg/L, while the acetate concentration was held at 100 μg/L. The number of cells produced in water samples was measured by the heterotrophic plate count (HPC) method using R2A agar. The result showed that the cell production of Pseudomonas fluorescens strain P17 and Spirillum strain NOX decreased with increasing salinity. Especially, the growth of Spirillum strain NOX was noticeably influenced by water salinity. To further observe the relation between acetate concentration and cell production in high salinity water, organic-free saline water samples were prepared by spiking NaCl in deionized (DI) water. The organic-free saline water samples were enriched with acetate of which concentration was varied to be 0–1,000 μg/L (as acetate). Also, P. fluorescens strain P17 was adjusted to high total dissolved solids (TDS) condition prior to being injected into the saline water samples. The result demonstrated that the amount of microorganisms increased with increasing acetate concentration. Although AOC measurement of saline water using Spirillum strain NOX seemed unacceptable, it was suggested that P. fluorescens strain P17 has the possibility to be used in measuring AOC in saline water. Moreover, the yield factor was altered as a result of reflecting salinity impact as the growth number of P. fluorescens strain P17 was unstable with high saline condition.