Pneumonia is an infection of the lungs, where the alveoli in the affected area are filled with pus and fluid. Although ventilated patients are at risk, not all ventilated patients develop pneumonia. This suggests that the sputum environment may possess antimicrobial activities. Despite the generally acknowledged importance of antimicrobial activity in protecting the human lung against infections, this has not been systematically assessed to date. Therefore, the objective of the present study was to measure antimicrobial activity in broncho-alveolar aspirate ('sputum") samples from patients in an intensive care unit (ICU) and to correlate the detected antimicrobial activity with antibiotic levels, the sputum microbiome, and the respective patients' characteristics. To this end, clinical metadata and sputum were collected from 53 mechanically ventilated ICU patients. The antimicrobial activity of sputum samples was tested against Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus anginosus. Here we show that sputa collected from different patients presented a high degree of variation in antimicrobial activity, which can be partially attributed to antibiotic therapy. The sputum microbiome, although potentially capable of producing antimicrobial agents, seemed to contribute in a minor way, if any, to the antimicrobial activity of sputum. Remarkably, despite its potentially protective effect, the level of antimicrobial activity in the investigated sputa correlated inversely with patient outcome, most likely because disease severity outweighed the beneficial antimicrobial activities.