<p>Biofilms are microbial communities of aggregated cells encased in extracellular matrix that are a pressing healthcare concern. Since biofilms have complex metabolic dynamics, in this study a new approach for studying biofilm metabolism is developed that employs optical-photothermal infrared (O-PTIR) spectroscopy imaging combined with ¹³C stable isotope probing and cryosectioning to track the carbon metabolism of cells at different depths of the biofilm. This approach demonstrated that metabolic gradients can be visualised using O-PTIR imaging, revealing a core of cells with low metabolic activity at the centre of the biofilm, with outer regions showing significantly higher metabolic activity. By incorporating the heavy stable isotope of carbon into bacterial biomass, we monitored the metabolic activity of gentamicin-resistant <i>Salmonella</i> Typhimurium within the biofilm structure upon exposure to various antibiotics. O-PTIR imaging revealed altered metabolic responses at various depths of the biofilm, with variations that depend on the bacterial antibiotic susceptibility profile.</p>