Textiles treated with antimicrobial agents are emerging as new strategies to reduce acquisition of healthcare-associated infections (HAIs). Essential to development/validation of these textiles are standard methods for the testing antimicrobial textile efficacy. Our laboratory has developed new testing methods, the fabric challenge assays, to recapitulate each transmission method and test the efficacy of antimicrobial textiles in a more "real world" simulation. 5 × 105 colony-forming units/ml (CFU/ml) MRSA suspensions were grown. 15 × 15 cm2 swatches of control, antimicrobial, hydrophobic barrier, and VTT003 fabric were inoculated with MRSA either by aerosol, splatter, or direct contact. Inoculated fabric was left at room temperature for 0, 30, or 60 min. Fabric was then transferred to buffer and shaken for 3 min at 400 rpm. A liquid suspension (0.1 ml) was then plated onto blood agar, grown overnight at 37°C, and colonies were counted. In the aerosol test, at 0 min, VTT003 significantly reduced MRSA by 78.52 ± 10.26% compared to control fabric. At 30 min, antimicrobial reduced MRSA levels by 91.48 ± 8.52%. In the splatter test, at 0 min, antimicrobial, hydrophobic barrier, and VTT003 fabrics reduced MRSA levels by 98.56 ± 1.44, 83.91 ± 13.16, and 100.00 ± 0.03%, respectively. At 30 min, hydrophobic barrier and VTT003 reduced levels by 82.63 ± 17.37 and 100.00 ± 0.00%, respectively. At 60 min, hydrophobic barrier and VTT003 abrogated MRSA levels. In the contact test, at 0 min, hydrophobic barrier and VTT003 reduced MRSA levels by 99.06 ± 0.94 and 97.08 ± 2.92%, respectively. At 30 min, hydrophobic barrier and VTT003 reduced MRSA levels by 100.00 ± 0.03 and 19.38 ± 19.38%, respectively. At 60 min, hydrophobic barrier and VTT003 abrogated MRSA levels. The fabric challenge assays are a novel method for evaluation of antimicrobial textile performance and should be considered in the development of standards and testing methods for all antimicrobial textiles intended for use in healthcare infection control strategies.