Healthcare-associated infections (HAIs) pose a significant public health threat in Canada, with over 200,000 cases annually. The Emergency Medical Services (EMS) sector, a critical part of the healthcare system, remains underexplored regarding its potential to harbor infection-causing pathogens. This study investigates the microbiome of EMS vehicles, focusing on pathogen detection using MinION® next-generation sequencing (NGS) and assessing the efficacy of current cleaning practices. The study involves sampling EMS vehicles at various locations and times to examine microbial variation and test the impact of cleaning products on microbial composition. A custom bioinformatics pipeline was developed to analyze sequencing data, identifying pathogen hotspots and providing insights into improving cleaning practices. Preliminary findings suggest that certain areas in the vehicles are more prone to microbial contamination, which could inform targeted cleaning interventions. This work also aims to simplify DNA sampling and processing, enabling EMS personnel to assess infection risks in the field with minimal training. Future efforts will expand to include broader EMS and public service sectors.