Background: Meningitis is a clinically important disease because of its high mortality and morbidity. The epidemiology of this disease has changed remarkably due to the introduction of pneumococcal vaccines and Haemophilus influenzae type b (Hib) conjugate vaccine. Therefore, it is required to continuously monitor and research the organisms isolated from cerebrospinal fluid (CSF) cultures. Methods: We analyzed trends of bacteria and fungi isolates obtained from CSF cultures between 1997 and 2016 in a tertiary care hospital according to year, month, gender, and age. Results: Out of a total of 38,450 samples, we identified 504 (1.3%) isolates. The isolation rate in the first tested decade (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006) ranged from 1.3% to 3.1%, while that in the second decade (2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016) ranged from 0.4% to 1.5%. The most common organisms was coagulase-negative staphylococci (CoNS) (31.9%), followed by Staphylococcus aureus (9.5%), Streptococcus pneumoniae (7.5%), Acinetobacter baumannii (5.8%), and Mycobacterium tuberculosis (5.8%).Monthly isolation rates were highest in May and July and lowest in February and December. Male to female ratio was 1.5:1. The isolation rates of S. pneumoniae, Enterococcus faecium, and Escherichia coli were similar in children and adults, but those of S. aureus, E. faecalis, A. baumannii, Pseudomonas aeruginosa, M. tuberculosis, and Cryptococcus neoformans were higher in adults than in children. Conclusion: During the last two decades, the isolation rate of CSF culture per year has decreased, with monthly isolation rates being highest in May and July. CoNS, S. aureus, and S. pneumoniae were most common in males, whereas CoNS, S. pneumoniae, and M. tuberculosis were most common in females. While Group B Streptococcus was most common in infants younger than 1 year, S. aureus and C. neoformans were more common in adults.