The detection of bacterial biofilms is essential for understanding their role in various fields, including medicine, industry, and environmental science. Traditional culture-based methods, despite their widespread use, have limitations in estimating biofilm biomass and providing comprehensive species composition information. Physical methods, such as direct observation and microscopy techniques, offer high-resolution visualization but may lack sensitivity. Chemical methods, including staining techniques and ATP-based assays, provide simple insights but face challenges in differentiating live and dead cells. Immunological methods, such as ELISA and immunofluorescence microscopy, offer high specificity but can be expensive and require expertise. Molecular methods like PCR, qPCR, and FISH enable specific detection but require optimization and cannot distinguish live from dead cells. Emerging technologies, such as biosensors, acoustic techniques, and mass spectrometry, hold promise for rapid, real-time monitoring but require further validation. Moving forward, considerations such as multimodal approaches, in situ detection methods, species-specific targeting, and automation and miniaturization will be crucial for advancing biofilm detection capabilities. By embracing these future directions, researchers can enhance our understanding of biofilm biology and develop targeted strategies for combating biofilm-related infections and environmental contamination.