Bacterial biofilms cause chronic diseases that are difficult to control. Since biofilm formation in space is well documented and planktonic cells become more resistant and virulent under modeled microgravity, it is important to determine the effect of this gravity condition on biofilms. Inclusion of glass microcarrier beads of appropriate dimensions and density with medium and inoculum, in vessels specially designed to permit ground-based investigations into aspects of low-shear modeled microgravity (LSMMG), facilitated these studies. Mathematical modeling of microcarrier behavior based on experimental conditions demonstrated that they satisfied the criteria for LSMMG conditions. Experimental observations confirmed that the microcarrier trajectory in the LSMMG vessel concurred with the predicted model. At 24 h, the LSMMG Escherichia coli biofilms were thicker than their normal-gravity counterparts and exhibited increased resistance to the general stressors salt and ethanol and to two antibiotics (penicillin and chloramphenicol). Biofilms of a mutant of E. coli, deficient in sigma(s), were impaired in developing LSMMG-conferred resistance to the general stressors but not to the antibiotics, indicating two separate pathways of LSMMG-conferred resistance.