The current chapter describes the potential of proteomics in understanding the drug resistance mechanisms of microbial biofilms. One of the most important and intriguing properties of microbial biofilms is their increased resistance to challenging environmental conditions. Microorganisms are capable of efficiently sensing harmful environmental conditions and adjusting their protein expression accordingly to obviate the challenges faced. The formation of biofilms involves the production of EPS by microorganisms, which constitutes a unique feature of the microbial biofilms. The analysis of biofilms using a time-course experiment would be an ideal way to capture the dynamic changes occurring during the biofilm development. The age of biofilms can affect the resistance phenomena observed. Studies on fungal biofilms agree with the hypothesis of increased antioxidative capacities as a mechanism for the higher antifungal resistance in biofilms. Studies on fungal biofilms agree with the hypothesis of increased antioxidative capacities as a mechanism for the higher antifungal resistance in biofilms.