Salmonella is a major food-borne pathogen able to persist in food processing environments because of its ability to form biofilms. A Salmonella enterica serotype Agona isolate from poultry (S24) was grown at 37°C in biofilms for up to 144 hours (H144) in attachment to polystyrene surfaces. Biofilm structures were examined at different stages in their development (H3, H24, H48, H72, H96 and H144) using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9), dead cells (propidium iodide), proteins (fluorescein isothiocyanate isomer I), lipids (DiD'oil), α-polysaccharides (concanavalin A, tetramethylrhodamine conjugate), and β-polysaccharides (calcofluor white M2R). Strain S24 developed a robust biofilm at H72 (biovolume of 166,852.5 ± 13,681.8 μm3 in the observation field of 16,078.2 μm2). The largest biovolume of live cells was also detected at H72 (128,110.3 ± 4,969.1 μm3), decreasing thereafter, which was probably owing to the detachment of cells prior to a new phase of colonization. The percentage of dead cells with regard to total cells in the biofilms increased throughout the incubation, ranging from 2.3 ± 1.1% (H24) to 44.2 ± 11.0% (H144). Proteins showed the greatest biovolume among the extracellular components within the biofilms, with values ranging from 1,295.1 ± 1,294.9 μm3 (H3) to 19,186.2 ± 8,536.0 μm3 (H96). Maximum biovolume values of 15,171.9 ± 660.7 μm3 (H48), 7,055.3 ± 4,415.2 μm3 (H144), and 2,548.6 ± 1,597.5 μm3 (H72) were observed for β-polysaccharides, α-polysaccharides and lipids, respectively. A strong (P < 0.01) positive correlation was found between the total biovolume of biofilm and the biovolume of live cells, proteins and β-polysaccharides, which may serve as useful markers of biofilm formation. The present work provides new insights into the formation of S. Agona biofilms. Our findings may contribute to the designing of reliable strategies for preventing and removing these bacterial communities.