Medical records of 54 patients whose cerebrospinal fluid (CSF) cultures were positive for Acinetobacter were retrospectively reviewed. In 34 (63.0%) patients, the Acinetobacter isolates were clinically insignificant. In the other 20 (37.0%) patients, presence of the organism was regarded as clinically significant and specific antimicrobial therapy was administered. Patients with clinically significant Acinetobacter in CSF more frequently resided in intensive care units (p = 0.011), had intracerebral hemorrhage (p = 0.012) or previous CNS infection (p = 0.003), had prior antibiotic exposure (p = 0.011), and had undergone neurosurgical procedures (p = 0.003). Their CSF was characteristic of neutrophilic pleocytosis, an elevated protein level, and a low CSF-to-serum glucose ratio. Apart from fever, no significant statistical differences existed in individual clinical symptoms and signs between the 2 groups of patients, while a combination of symptoms efficiently differentiated clinically significant and insignificant Acinetobacter. The finding of multiple CSF specimens positive for Acinetobacter was highly suggestive of an active CNS infection (p<0.001). The high ratio of clinically insignificant Acinetobacter CSF isolates highlights the importance of careful judgment when the organism is cultured from CSF. Antimicrobial therapy must be initiated promptly in patients with identified risk factors, while unnecessary treatment should be avoided in those without them.