Lactobacillus salivarius belongs to the microbiota of human oral cavity and gastrointestinal tract, as well as of bird and pig intestines. Probiotic activity of various L. salivarius strains has been recently extensively investigated. Production of exopolysaccharides and formation of biofilms as a mechanism providing for resistance to unfavorable factors are also of interest. The goal of this work was to assess the efficiency of microbiological methods for analysis of bacterial concentrations in the cultures of L. salivarius strain NBR2. Samples of lactobacteria grown in liquid medium were collected at equal intervals. The parameters determined were the number of colony-forming units (CFU/mL), share of dead cells by the membrane permeabilization test (LIVE/DEAD) using flow cytometry and fluorescence microscopy, optical density at 595 nm, and pH. After 10 h of cultivation, formation of aggregates commenced, which consisted mainly of living cells and were detected throughout the experiment (30 h). This resulted in underestimation of bacterial abundance determined by plating (CFU/mL). Optical density of the culture and the share of dead cells determined by the LIVE/DEAD method are more reliable criteria of growth of the statically developing L. salivarius culture, which is prone to formation of biofilms and cell aggregates.