Optofluidic sensors based on periodic arrays of subwavelength apertures that support surface plasmon resonance can be employed as both optical sensors and nanofluidic structures. In flow-through operation, the nanoapertures experience pressure differences across the substrate in which they are fabricated, which imposes the risk for structural failure. This work presents an investigation of the deflection and structural stability of nanohole array-based optofluidic sensors operating in flow-through mode. The analysis was approached using experiments, simulations via finite element method, and established theoretical models. The results depict that certain areas of the sensor deflect under pressure, with some regions suffering high mechanical stress. The offset in the deflection values between theoretical models and actual experimental values is overturned when only the effective area of the substrate, of 450 µm, is considered. Experimental, theoretical, and simulation results suggest that the periodic nanostructures can safely operate under trans-membrane pressures of up to 20 psi, which induce deflections of up to ~20 μm.