Introduction: Bronchoscopy is a valuable diagnostic tool and has a significant clinical impact on the management of pneumonia, especially ventilator-associated pneumonia in ICU patients who fail to respond to standard, guideline-based therapy. The investigation of bronchial alveolar lavage (BAL) specimens involves an extensive laboratory work. Currently, there are very few published studies evaluating the diagnostic benefit of collecting multiple BAL specimens during bronchoscopy. These redundant specimens result in a significant workload increase for the diagnostic microbiology laboratory. Objective: To investigate the optimal number of specimens for bacteriology, virology, mycology, Pneumocystis jirovecii (PJP) and Legionella penumophila specimens in order to optimize the utility of BAL specimens with the aim of minimizing harm to the patient and optimizing resource utilization for the laboratory. Method: BAL specimens collected at an academic institution in Southern Ontario were reviewed retrospectively over a 15 month period for bacteriology, 16 months for virology, one year for mycology, and three years for L. pneumophila and P. jirovecii. Results: One thousand sixty-three BAL specimens were ordered for bacterial culture, yielding positive results in 45.5%. Among them, a concordance rate of 97.1% was found between two or more specimens acquired from different lung lobes. The concordance rate of multiple virology samples was 98.6% among patients in whom 2 specimens were collected per procedure, and 100% among those with 3 specimens per procedure. To study whether one specimen is sufficient for the detection of filamentous fungi, we reviewed 43 BAL samples which grew Aspergillus fumigatus between. A concordance rate of only 60% was found between two specimens obtained from different lung lobes. A concordance rate of 100% was found among multiple specimens ordered for L. pneumophila and P. jirovecii with positivity rates of 0.14% and 0.92% respectively. Conclusion: We recommend a single specimen per BAL be sent from the most purulent lung segment for bacteriology and viral PCR. Single specimens may also be appropriate for L. pneumophila and P. jirovecii, however further study is needed. Multiple specimens should be submitted for mycology investigations. By eliminating duplicate specimens laboratory utilization can be optimized and patient morbidity may be decreased.