Many bacterial species readily develop biofilms that act as a protective matrix against external challenge, e.g., from antimicrobial treatment. Therefore, biofilms are often responsible for persistent and recurring infections. Established methods for studying biofilms are either destructive or focus on the biofilm’s surface. A non-destructive method that is sensitive to the underside of the biofilm is highly desirable, as it allows studying the penetration of antibiotics through the film. Here, we demonstrate that the high surface sensitivity of resonant hyperspectral imaging provides this capability. The method allows us to monitor the early stages of Escherichia coli biofilm formation, cell attachment and microcolony formation, in-situ and in real-time. We study the response of the biofilm to a number of different antibiotics and verify our observations using confocal microscopy. Based on this ability to closely monitor the surface-bound cells, resonant hyperspectral imaging gives new insights into the antimicrobial resistance of biofilms.