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Background: Methicillin-resistant Staphylococcus aureus (MRSA) can be found in emergency medical services (EMS) ambulances. This poses an occupational risk and patient safety hazard. Screening for environmental contamination is often not performed due to limited resources and logistical challenges. This study’s objective was to compare traditional screening of individual surfaces versus “pooled sampling” to efficiently identify contamination. Methods: A cross-sectional study, conducted among 145 Ohio EMS ambulances from 84 agencies, tested a novel pooled sampling methodology to detect MRSA contaminated ambulances. For ambulances screened using pooled sampling, 3 samples were collected within each ambulance. Pool One included cabinets, doorways, and ceiling bar. Pool Two included cot, seats, and backboard. Pool Three included steering wheel, kits, and clipboard. For ambulances screened with the traditional detection technique, each of the 9 aforementioned surfaces were sampled individually. Descriptive statistics and unadjusted and adjusted odds ratios (OR) were calculated to compare the 2 methods. Results: Forty-seven of 145 ambulances (32.4%) had at least 1 of the 9 locations contaminated with MRSA. When comparing the 2 screening methodologies, no significant difference was observed regarding the overall detection of MRSA contaminated ambulances (24/60 [40%] versus 23/85 [27.6%], P value: 0.1000). This indicates that pooled sampling appears as an efficient method for identification of MRSA contaminated ambulances. Conclusion: One-third of Ohio ambulances had MRSA contamination in this study. Therefore, an efficient methodology to identify contaminated ambulances with hazardous pathogens is incredibly valuable. Pooling can help save resources and simplify sampling logistics, all which could positively impact infection control practices in emergency medical services.