Antibiotic susceptibility testing is widely performed in clinical microbiology labs. The disk diffusion method is the gold standard for confirming the susceptibility of bacteria. Using this method, introduced by Bauer and Kirby in 1956, a standardized bacterial suspension is prepared and inoculated onto solidified agar and an antibiotic-treated paper (disk) is tapped on the inoculated plate. The disc containing the antibiotic is allowed to diffuse through the solidified agar, resulting in the formation of an inhibition zone after the overnight incubation at 35 °C. The size of the inhibition zone formed around the paper disc is measured and the assay generally takes 16–24 h. Assessing the inhibition zone using this assay gives a relatively clear picture of the antibiotic susceptibility of the bacteria, yet it is unclear if the antibiotic also impacts the extracellular matrix in which the bacterial cells are embedded. As a result, patients may receive an insufficient antibiotic dose and treatment, putting them at risk of recurrent infections. Furthermore, inappropriate antibiotic use contributes to the development of antimicrobial resistance. In a recent study, published in the Journal Biofilm, researchers at AIMES located at Karolinska Institutet in Stockholm, Sweden, employed an EbbaBiolight-based biofilm assay to shed light on the interaction between antibiotics and the extracellular matrix formed by Salmonella colonies on agar plates. The agar was supplemented with EbbaBiolight 680 and, in accordance with the disk-diffusion assay, antibiotic discs were used to assess the antibiofilm efficiency of ten clinically relevant antibiotics for Salmonella. While the majority of antibiotics demonstrated the ability to decrease colony growth, only 4 antibiotics exhibited the capacity to inhibit the formation of the curli-based extracellular matrix. Notably, colonies lacking curli were more susceptible to antibiotics than colonies with intact curli synthesis. This suggests that antibiotics that can reduce curli production have the potential to enhance the sensitivity of colonies to other antibiotics, making them valuable candidates for inclusion in combination therapies for more effective treatment of biofilm infections. This study emphasizes the need for biofilm-specific treatment strategies and readily available tools for their development. In this context, the EbbaBiolight molecules can provide an easy to use, readily available tool for studying biofilm formation and evaluating the effects of antimicrobials.