Objective To evaluate the effectiveness of filter membranes made from different materials in monitoring the health status of rodents in barrier environment facilities by investigating their microbial capture performance. Methods Pasteurella pneumotropica (Pp) and Staphylococcus aureus (Sa) were used as representative strains to simulate the process of microbial capture by filter membranes under laboratory conditions. The microbial capture effectiveness of five self-selected filter membranes (M1, M2, M3, M4, and M5) with adsorption and breathability properties and a commercial filter membrane (T1) were comprehensively evaluated based on captured dust mass, minimum detection limit, and differences in Ct values obtained through fluorescence quantitative PCR detection. The best-performing self-selected filter membrane was placed in the ventilation ducts of cage racks within the barrier facility, with sentinel mice in corresponding cage racks as the control group. Staphylococcus epidermidis and Escherichia coli were used as indicator bacteria to calculate the positive detection rate and coincidence rate, thereby exploring the feasibility of using microbial capture filter membranes to monitor the health status of experimental animals in barrier facilities. Results In terms of the captured dust mass, the self-selected filter membrane M3 (non-woven filter membrane with a diameter of 0.1 um)；showed a capture effectiveness second only to T1, with a capture mass of 0.126 g. For Sa, all filter membranes except M4 had a minimum detection limit of 10² CFU/g. For Pp, the minimum detection limit for all filter membranes was 10² CFU/g. However, the Ct value of the quantitative fluorescence PCR amplification results for M3 was significantly lower than that of other materials, indicating that M3 had the best capture performance among the five self-selected materials. In the filter detection verification experiment, the positive detection rate of Staphylococcus epidermidis in sentinel mouse feces and M3 was 50.00% (6/12) and 58.33% (7/12), respectively, with a coincidence rate of 92%. The positive detection rate of Escherichia coli in both sentinel mouse feces and M3 was 50.00% (6/12), with a coincidence rate of 100%. Conclusion Among the 5 self-selected filter membranes, M3 exhibits the best capturing performance. Within the barrier environment facilities, M3 outperforms sentinel mice in monitoring Staphylococcus epidermidis. Therefore, non-woven filter membrane with a diameter of 0.1 um； can be used as the material for microbial capture filter membranes, providing valuable insights for the selection and application of microbial capture filter membranes used in PCR monitoring of cage exhaust air dust.