Background: To properly clean and disinfect hospital mattresses, bed manufacturers recommend a 3- to 6-step process to remove all pathogenic bacteria. An alternative is to use a removable barrier on the mattress, which is laundered after each use. The current study was to determine efficacy of a commercial laundry process in eliminating Clostridioides difficile (C diff) spores, Mycobacterium terrae (M terrae), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P aeruginosa), Klebsiella pneumoniae (K pneumoniae), and Escherichia coli (E coli) from a barrier. Methods: A test barrier received 3 unique microbial suspensions in separate locations, each suspension having a known quantity of specific microorganisms: C diff spores, M terrae, and a mixed suspension of MRSA, S aureus, P aeruginosa, K pneumoniae, and E coli. A wash load contained the test barrier and 11 additional ballast barriers. Various soils were spread onto the barriers to simulate heavy soiling that may occur in a wash load: Each barrier received a small amount of mixed soil, 50% received urine, 25% received blood, and 25% received a large amount of additional mixed soil. The load was laundered using 71°C (160°F) water, detergent, and chlorine bleach, with final drying at 71°C (160°F). After laundering, remaining colony-forming units (CFUs) of each microorganism were counted at the applied locations. Each test was replicated 3 times. Industry-accepted methods were used to produce suspensions, apply inoculum, and recover organisms after laundering. Results: Before laundering, test barriers contained at least 7.0 log10 cfu/mL of each microorganism distributed over 103 cm2. After laundering, in all cases, no residual CFUs were detected over the test area, resulting in greater than 6.0 log10 reductions for every organism. ( P < .05). Conclusions: Under extreme test conditions including the presence of soil, the laundry process removed all detectable pathogenic bacteria and spores from the barrier.