This study aimed to evaluate the effect of low salinity on the water quality, microbial flocs composition and performance of Litopenaeus vannamei juveniles reared over 40 days in a zero-water-exchange super-intensive system at 0, 2, 4 and 25 g L−1. At 0 g L−1, the mortality was total at the 26th day, and consequently, these salinity data were not included in the statistical analysis. Among the water quality parameters, only pH and the total suspended solids concentration were significantly influenced by salinity. However, a trend towards intensification of the nitrification processes was observed as the salinity increased, with the lowest ammonia and the highest nitrite and nitrate concentrations found at 25 g L−1. The concentrations of ciliates and flagellates diminished and increased, respectively, with the increase in salinity. Diatoms predominated at 25 g L−1, whereas at 2 and 4 g L−1, chlorophytes were more abundant. Microbial floc crude protein content was reduced with the increase in salinity, whereas ash content demonstrated the inverse trend. The best overall growth performance and survival were observed at 25 g L−1. However, satisfactory productivity was also found at 4 g L−1, suggesting the viability of rearing L. vannamei at low salinity under zero-water-exchange conditions.