Abstract Background Central nervous system (CNS) infections are associated with significant adverse clinical outcomes. Approximately 50% of all CNS infections are idiopathic. The impact of the Biofire® FAME in Colombia is unknown. Methods A retrospective study of all adults with CNS infections in one tertiary hospital in Colombia. The cohort was divided into two time periods: before and after the implementation of the Biofire® FAME panel in May 2016. Etiologies, management decisions, and outcomes were compared between both groups. Data analysis was done with Stata® version 14. Results A total of 76 patients were enrolled. The majority of the patients (72.4%) were male with a median age of 40.9 years. The most common comorbidity was Human immunodeficiency virus (HIV) infection (47.4%); 54% were admitted to the intensive care unit (ICU) and 29% died. Out of the 76 patients, 33 and 43 were enrolled in the pre- and post-Biofire FAME testing periods, respectively. A diagnosis was made more commonly in the post-Biofire time period (13/33;39% vs. 27/43;63%, P < 0.05). The etiologies in the pre-Biofire group were Cryptococcus neoformans (6); bacterial meningitis (5) and tuberculosis (1) and varicella zoster (1); and the etiologies in the post-Biofire group were Cryptococcus neoformans (7), cytomegalovirus (CMV) (4); bacterial meningitis (4), tuberculosis (3), syphilis (3), ADEM (3), CNS vasculitis (1), and neurocysticercosis (1). The Biofire FAME panel was positive in 14 patients: C. neoformans (6), CMV (3), S. pneumoniae (2), Herpes simplex virus type 2 (2), H. influenzae (1) and N. meningitidis (1). In 12 out of those 14 patient (85%), a change in therapy occurred due to the FAME results (5 started therapy, four stopped therapy and threehad a change in therapy). Furthermore, patients in the post-Biofire FAME period had a reduction in the empirical use of vancomycin, meropenem and cefepime (P < 0.05). The Biofire FAME panel missed identifying one patient with Cryptococcus neoformans and one with CMV. Conclusion The introduction of the Biofire FAME panel in a hospital with high HIV prevalence in Colombia has increased the yield of pathogens identified and have reduced the use of empirical antibiotic therapy. The Biofire FAME failed to identify one case of Cryptococcus neoformans and one due to CMV. Disclosures R. Hasbun, Biofire: Speaker’s Bureau, Speaker honorarium Biomeriaux: Consultant, Consulting fee.