Glass fiber filters are widely used in ventilation and environmental control systems of buildings and transportation vehicles to removeairborne particulate pollutants to keep healthy indoor air quality (IAQ). Abundant particles with organic components and appropriate airenvironment conditions make general ventilation air filters as potential sites for microbial residence and growth. Secondary bioaerosolcontamination caused by microbial growth on air filters becomes a threat to IAQ. Significant associations between bioaerosols levels and airenvironmental conditions make it important for us to study the effects of environmental factors on microorganism growth. We designed a test systemto put microorganisms on the filter media and cultivate the media under different conditions for several days. A thermostat water bath and ahumidifier were used in the test system to control air temperature and relative humidity. Serratia marcescens and Bacillus subtilis were used asexperimental tracer bacteria. The microorganism counting numbers on glass fiber filter media declined nonlinear when nutrient quantity or relativehumidity declined. This study shows that air temperature, relative humidity and water content on the filter media have great influence on microbialgrowth on glass fiber filter media.