The COVID-19 pandemic has highlighted the significant infection risks posed by aerosol generating procedures (AGPs). We developed a hood that covers the patient’s respiratory area, incorporating a negative pressure system to contain aerosols. This study analyzed the movement and containment of aerosols within a developed negative pressure isolation chamber. Using particle image velocimetry (PIV) technology, in the optimized design, the characteristics of aerosols were analyzed under both negative and non-negative pressure conditions. The results demonstrated that in the absence of negative pressure, droplets dispersed widely, with diffusion angles ranging from 26.9° to 34.2°, significantly increasing the risk of external leakage. When negative pressure was applied, the diffusion angles narrowed to 20.0–35.1° and inward airflow effectively directed droplets away from the chamber boundary, preventing external dispersion. Additionally, sensor data measuring particle concentrations confirmed that droplets smaller than 10 µm were fully contained under negative pressure, strongly supporting the chamber’s effectiveness. The strong agreement between PIV flow patterns and sensor measurements underscores the reliability of the experimental methodology. These findings highlight the chamber’s ability to suppress external leakage while offering superior flexibility and portability compared to conventional isolation systems, making it ideal for emergency responses, mobile healthcare units, and large-scale infectious disease outbreaks.