Ziehl-Neelsen (ZN) microscopy is the primary method for acid-fast bacilli examination in resource-limited settings, including Thailand. Despite its considerably improved diagnostic performance, conventional fluorescent microscopy (FM) is rarely used due to its perceived high cost. An evaluation in Thailand found that the total cost of FM operated in the National Tuberculosis Reference Laboratory (NTRL) in Bangkok, Thailand, is similar to that of ZN performed in the NTRL and in four regional Thai laboratories. FM is therefore a cost-effective alternative to ZN in resource-limited settings.