An improved method for direct fungal identification and enumeration in air and surface samples was developed for use at the Department of Energy’s Savannah River Site (SRS), Aiken, SC. Direct microscopic examination of fungal hyphae and conidia is often difficult for indoor samples due to debris, including pollen and fluorescent textile fibers. Therefore, a staining method incorporating FUN-1 (Molecular Probes, Eugene, OR), Fluorescent Brightener 28 (Sigma Chemical Co., St. Louis, MO), and potassium hydroxide was developed to directly examine microorganisms in air and physical samples. The sampling included environmental samples from several buildings using the Andersen 6-Stage Viable Particle-Sizing Air Sampler (Smyrna, Georgia), and direct surface sampling were fungal growth was suspected. Split samples showed the new staining method was more effective in detecting and distinguishing fungal structures collected during sampling and also enhanced clarity of structures of fungal isolates. Application of this technique has increased the speed and sensitivity of fungi detection for workplace monitoring. This method was applied to workspace assessments and has increased understanding of the relationships between fungal growth on surfaces, airborne fungi, environmental factors and overall workspace assessment.