In this study, four different HVAC filters were tested to evaluate the filter layer structure effect on the Most Penetrating Particle Size. Each filter consisted of two melt-blown (MB) layers and a thermal bond (TB) layer. The MB layers were constructed with fibers with diameters of 2 μm and 5 μm diameter while the TB layer exhibited a fiber diameter of 37 μm. The positions of the three layers (MB2, MB5, and TB) were varied along the air flow direction, and four different filters were considered as follows: Filter A (MB2 → TB → MB5), Filter B (MB5 → TB → MB2), Filter C (TB → MB2 → MB5), and Filter D (TB → MB5 → MB2). When particle loading was absent (clean filter), pressure drops and collection efficiencies were almost identical for the filters, thereby indicating that the filtration performance did not significantly change with the filter layer arrangement. However, particle loading test results showed that pressure drop and most penetrating particle size (MPPS) trends depended on the location of the MB2 layer. For Filter D, the pressure drop and the decreasing rate of the MPPS were the lowest. The pressure drop increased from 3.5 to 9.4 mmH2O and the MPPS decreased from 270 to 160 nm, when the particle loading increased from 0 to 1.55 g/m2.