Biofilms represent a key survival strategy, providing bacteria with effective protection against abiotic and biotic stress. In the laboratory, Bacillus subtilis subsp. spizizenii forms a biofilm at the liquid–air interface, the matrix of which is primarily composed of polysaccharides, proteins, and nucleic acids. The aims of this study are as follows: 1) to evaluate how the chemical composition of the matrix and the stability of the biofilm are modified when the bacteria grow using either glucose or glycerol as carbon sources; 2) to study the effect of temperature on biofilm production and planktonic growth. The bacteria were cultured in a saline medium with 55 mM L-glutamic acid and 1% glucose or glycerol at different temperatures. Biofilm was produced under static conditions and planktonic cells by shaking at 150 rpm. Biofilms were analyzed by high-resolution chromatography. In the presence of glucose, polysaccharide monomers at the biofilm matrix showed substitutions that conferred acidic properties. This biofilm disintegrated after 5 days at 20 °C and 15 days at 4 °C. In contrast, when cultured with glycerol, the polysaccharide sugars were neutral, and the biofilm disintegrated after 20 days at 20 °C and 60 days at 4 °C; visually, this biofilm was more compact. Planktonic growth of B. subtilis was highest at 45 °C, whereas the maximum biofilm formation (1.2 mg/mL of culture) was achieved between 30 °C and 37 °C. In the static cultures, the planktonic form of B. subtillis disappeared within 24 hours after biofilm synthesis. In conclusion, the biofilms obtained from different carbon sources show differences, suggesting that the substituents of the polysaccharide monomers might be crucial for their structural properties. Moreover, in cases where the biofilm was formed at the liquid–air interface, the planktonic cells developed in this culture migrated toward the biofilm and as a result, the medium appeared clear; this was likely due to to nutrient depletion, which does not support planktonic growth.