Objectives/Hypothesis: In critically ill patients, the occurrence of artificial ventilation-acquired sinus disease is common. A possible sinus bacterial infection is occult and is combined with diagnostic difficulties. Culture as a method has limited capacity to verify the presence of bacteria and leaves unanswered the question of a possible infective agent because bacteria are difficult to grow when killed or suppressed by current antibiotic therapy. Hitherto unidentified micro-organisms are also possible in the microenvironments of maxillary sinuses. Study Design: Prospective case series. Methods: Twenty maxillary sinus samples (17 aspirates and 3 lavages) from nine critically ill patients with possible infectious disease were investigated by broad-range 16S rRNA polymerase chain reaction followed by sequencing. These results were compared with the previous culture results from gingiva (passage route), maxillary sinus absorption, and mucosa samples. Results: The contaminations were rare (2 of 20) and corresponded well to culture results. One previously undiagnosed bacterium was found. Two aspirates were negative on both culture and polymerase chain reaction, whereas the corresponding maxillary sinus mucosal cultures had been positive. Conclusions: Applying a lowcontamination maxillary sinuses sampling technique makes 16S rRNA sequencing clinically useful. The polymerase chain reaction and culture results were generally comparable. However, by polymerase chain reaction, bacteria were found that were missed by culturing. Some aspirates were free of bacterial remnants on 16S rRNA polymerase chain reaction, but corresponding mucosal cultures were positive. This could indicate that infection is induced within the tissue. It also indicates that infectious agents introduced into the sinuses may have routes other than the ostium. Further clinical use of 16S rRNA sequencing is required to enlarge our knowledge in applied microbiology and paranasal sinus disease.