Biofilms are ubiquitous and notoriously difficult to eradicate and control, complicating human infections, industrial and agricultural biofouling. Current biofilm studies are commonly performed with the biofilm cultured on mono-interface and generally have neglected to consider more realistic biofilm, where diverse interfaces are involved. In our study, a reusable dual-chamber microreactor with interchangeable membranes was developed to establish multiple interfaces for biofilm culture and test. Protocol for culturing Pseudomonas aeruginosa (PAO1) on the air-liquid interface (ALI) and liquid-liquid interface (LLI) under static environmental conditions for 48h was optimized using this novel device. This study shows that LLI model biofilms are more susceptible to physical disruption compared to ALI model biofilm. SEM images revealed a unique ‘mushroom-shaped’ microcolonies morphological feature, which is more distinct on ALI biofilms than LLI. Furthermore, the study showed that ALI and LLI biofilms produced a similar amount of extracellular polymeric substances (EPS). As differences in biofilm structure and properties may lead to different outcomes when using the same eradication approaches, the antimicrobial effect of an antibiotic, Ciprofloxacin (CIP), was chosen to test the susceptibility of 48h-old ALI and LLI biofilms. Our results show that the minimum eradication concentration (MBCE) of CIP using our dual-chamber device reached 1600μg/ml, which is significantly higher than the conventional microtiter plate method (64μg/ml). The results highlight the importance of having a model that can closely mimic in-vivo conditions to develop more effective biofilm management strategies.