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• Crystal violet staining for air–liquid biofilms can show false positives. • Novel reflection-based method detects pellicle formation in a well plate. • Pendant droplet tests shown to have significant potential for biofilm studies. In food safety and food quality, biofilm research is of great importance for mitigating food-borne pathogens in food processing environments. To supplement the traditional staining techniques for biofilm characterization, we introduce several non-traditional imaging methods for detecting biofilm attachment to the solid–liquid and air–liquid interfaces. For strains of Pseudomonas aeruginosa (the positive control), Acinetobacter baumanii , Listeria monocytogenes and Salmonella enterica , the traditional crystal violet assay showed evidence of biofilm attachment to the well plate base as well as inferred the presence of an air–liquid biofilm attached on the upper well walls where the meniscus was present. However, air–liquid biofilms and solid-surface-attached biofilms were not detected for all of these strains using the non-traditional imaging methods. For L. monocytogenes , we were unable to detect biofilms at a particle-laden, air–liquid interface as evidenced through microscopy, which contradicts the meniscus staining test and suggests that the coffee-ring effect may lead to false positives when using meniscus staining. Furthermore, when L. monocytogenes was cultivated in a pendant droplet in air, only microbial sediment at the droplet apex was observed without any apparent bacterial colonization of the droplet surface. All other strains showed clear evidence of air–liquid biofilms at the air–liquid interface of a pendant droplet. To non-invasively detect if and when air–liquid pellicles form in a well plate, we also present a novel in situ reflection assay that demonstrates the capacity to do this quantitatively. [ABSTRACT FROM AUTHOR]