Antibiotics, no doubt, have revolutionized the healthcare settings; the recent resurgence of its decreased sensitivity due to the increased resistance profile exhibited by majority of pathogenic microorganisms become a serious public issue. The increased resistance profile of pathogenic microorganisms could be attributed to the ability to form recalcitrant biofilms which is basically an adaptive survival strategy against environmental stress. Due to biofilm formation, a significant implication is observed in terms of healthcare settings as well as socioeconomic values. In this regard, it is important to monitor the biofilm pathophysiological progression with real-time detection and diagnosis. To achieve this, omics-based tools, particularly transcriptomics analysis, have revolutionized the diagnostic values of biofilm mechanics. In addition, the transcriptomics analysis could also be instrumental in spatial design and development of improved therapeutic agents against chronic microbial infections. In this chapter, we provided an insight into the transcriptomics-based approaches to understand the biofilm pathophysiology with an aim to real-time diagnosis and therapeutic interventions in the management of biofilm-associated chronic infections and drug resistance phenomena. This chapter also emphasized on the role of several therapeutic agents in the downregulation of several differentially expressed genes (DEGs) linked with biofilm pathophysiology, and how these DEGs could be explored in the regulation of biofilm-mediated virulence and antibiotic resistance phenomena.