Fabrics act as fomites for microorganisms, thereby playing a significant role in infection transmission, especially in the healthcare and hospitality sectors. This study aimed to examine the biofilm formation ability of four nosocomial infection-causing bacteria (Acinetobacter calcoaceticus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) on cotton, polyester, polyester-cotton blend, silk, wool, viscose, and nylon, used frequently in the healthcare sector, by qualitative and quantitative methods. The impact of temperature, pH, and relative humidity(RH) on biofilm formation was also assessed. P. aeruginosa and S. aureus were strong biofilm producers, while E. coli produced weak biofilm. Wool (maximum roughness) showed the highest bacterial load, while silk (lowest roughness) showed the least. P. aeruginosa exhibited a higher load on all fabrics, than other test bacteria. Extracellular polymeric substances were characterized by infrared spectroscopy. Roughness of biofilms was assessed by atomic force microscopy. For biofilm formation, optimum temperature, pH, and RH were 30°C, 7.0, and 62%, respectively. MgCl2 and CaCl2 were the most effective in removing bacterial biofilm. In conclusion, biofilm formation was observed to be influenced by the type of fabric, bacteria, and environmental conditions. Implementing recommended guidelines for the effective disinfection of fabrics is crucial to curb the risk of nosocomial infections. In addition, designing modified healthcare fabrics that inhibit pathogen load could be an effective method to mitigate the transmission of infections.