Background Patients admitted to intensive care units are frequently exposed to pathogenic microorganisms present in their environment. Exposure to these microbes may lead to the development of hospital-acquired infections that complicate the illness and may be fatal. Amoeba-associated microorganisms (AAMs) are frequently isolated from hospital water networks and are reported to be associated to cases of community and hospital-acquired pneumonia. Methodology/Principal Findings We used a multiplexed immunofluorescence assay to test for the presence of antibodies against AAMs in sera of intensive care unit (ICU) pneumonia patients and compared to patients at the admission to the ICU (controls). Our results show that some AAMs may be more frequently detected in patients who had hospital-acquired pneumonia than in controls, whereas other AAMs are ubiquitously detected. However, ICU patients seem to exhibit increasing immune response to AAMs when the ICU stay is prolonged. Moreover, concomitant antibodies responses against seven different microorganisms (5 Rhizobiales, Balneatrix alpica, and Mimivirus) were observed in the serum of patients that had a prolonged ICU stay. Conclusions/Significance Our work partially confirms the results of previous studies, which show that ICU patients would be exposed to water amoeba-associated microorganisms, and provides information about the magnitude of AAM infection in ICU patients, especially patients that have a prolonged ICU stay. However, the incidence of this exposure on the development of pneumonia remains to assess.