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Purpose Cytomegalovirus (CMV) reactivation, a significant cause of morbidity and mortality in immunosuppression, may affect “immunocompetent” seropositive critically ill patients. The aim of this prospective, observational study was to define the incidence, risk factors, and the association with morbidity and mortality of CMV reactivation in a general population of critically ill immunocompetent patients. We also studied the relationship between reactivation and patients' inflammatory response, as expressed by cytokine levels and stress up-regulation by salivary cortisol. Methods This study included mechanically ventilated CMV-seropositive patients. A quantitative real-time polymerase chain reaction (PCR) was performed for CMV plasma DNAemia determination, upon intensive care unit (ICU) admission and weekly thereafter until day 28. Cytomegalovirus reactivation was defined as CMV plasma DNAemia greater than or equal to 500 copies/mL. Upon ICU admission, interferon γ, interleukin (IL) 10, IL-17A, IL-2, IL-6, and tumor necrosis factor α were quantified in plasma, and morning saliva was obtained to measure cortisol. Disease severity was assessed by Acute Physiology and Chronic Health Evaluation II score, whereas the degree of organ dysfunction was quantified by Sequential Organ Failure Assessment score. Mortality, duration of mechanical ventilation, and ICU length of stay were recorded. Results During the study period, 80 (51 men) patients with a median age of 63 years fulfilled the inclusion criteria. Reactivation of CMV occurred in 11 patients (13.75%). Median day of reactivation was day 7 post ICU admission. Total number of red blood cell units transfused (odds ratio [OR], 1.50; confidence interval [CI], 1.06-2.13; P = .02) and C-reactive protein levels upon ICU admission (OR, 1.01; CI, 1.00-1.02; P = .02) were independently associated with CMV reactivation. High IL-10 was marginally related to reactivation (P = .06). Sequential Organ Failure Assessment scores were higher in the group with CMV reactivation compared with patients without reactivation during the entire 28-day observation period (P < .006). Salivary cortisol, mortality, length of ICU stay, and duration of mechanical ventilation were similar in the 2 groups. Conclusions Cytomegalovirus reactivation occurred in 13.75% of critically ill, immunocompetent patients. The degree of inflammation and the total number of transfused red blood cells units constituted risk factors. Cytomegalovirus reactivation was associated with more severe of organ dysfunction, but not with a worse clinical outcome.