Abstract Background Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in microbial identification. This study evaluated the performance of MALDI-TOF MS and investigated the economic and medical impact of MALDI-TOF MS implementation. Methods A total of 12,202 clinical isolates collected from April to September 2013 were identified using MALDI-TOF MS, and the success rates in identifying isolates were analyzed. The differences in the processing time, cost of consumables, weight of waste, and clinical impact between MALDI-TOF MS and biochemical reaction were compared. Results MALDI-TOF MS successfully identified 96% of 12,202 isolates, including 96.8% of 10,502 aerobes, 90.5% of 1481 anaerobes, 93.8% of 81 yeasts, and 90.6% of 138 nontuberculous mycobacteria at the genus level. By using MALDI-TOF MS, the processing time for aerobes decreased from 32.5 hours to 4.1 hours, and that for anaerobes decreased from 71.5 hours to 46 hours. For detection of aerobes and anaerobes, the cost of consumables was estimated to decrease by US$0.9 per isolate, thus saving US$94,500 in total annual isolation. Furthermore, the weight of waste decreased six-fold, resulting in a reduction of 350 kg/month or 4.2 tons/year. MALDI-TOF MS also increased the percentage of correct antibiotics treatment for Escherichia coli and Klebsiella pneumonia from 56.1% to 75% and shortened the initiation time of the correct antibiotic action from 3.3 hours to 2.5 hours. Conclusions MALDI-TOF MS is a rapid, reliable, economical, and environmentally friendly method for routine microbial identification and may contribute to early appropriate antibiotic treatment in clinical settings.