Microbial biofilms are structured communities of microorganisms embedded in a self-produced extracellular matrix, adhering to surfaces. These biofilms enhance bacterial resistance to antibiotics, immune responses, and environmental stress. Current microscopy techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and fluorescence microscopy, are commonly used to visualize and differentiate biofilms. However, their high cost and complexity often render them impractical. In contrast, simpler methods like crystal violet and Congo red staining are limited in distinguishing bacterial cells from the biofilm matrix. This study introduces a cost-effective, dual-staining method using Maneval’s stain to visualize and differentiate microbial biofilms. It requires only basic equipment and minimal reagents, making it ideal for routine use in clinical diagnosis and microbial research.