Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 4 2026 08 04 473 481 Beena Antony Department of Microbiology, Fr. Muller Medical College, Karnataka, India Sherin Justin Department of Microbiology, Fr. Muller Medical College, Karnataka, India Shruthi Bagambila Fr. Muller Research Center, Mangalore, Karnataka, India Jane D’Souza Fr. Muller Research Center, Mangalore, Karnataka, India Rhea Pinto Fr. Muller Research Center, Mangalore, Karnataka, India 2025 11 26 2026 06 26 Background and Objectives: MALDI-TOF MS offers a unique combination of flexibility, accuracy, automation and has found benefit in many fields of biological research. Here we investigated MALDI-TOF analysis for the identification of anaerobic and microaerophilic bacteria isolated from clinical specimen and also compared the results with conventional biochemical characterization. Materials and Methods: The present study was conducted for a duration of 2 years and had obtained Ethical clearance. Bacteria obtained from 1514 specimens were subjected to MALDI-TOF in comparison with conventional techniques. MALDI-TOF was done by Extended Direct Transfer technique after a preparatory protein extraction. Results: Of the 957 isolates of anaerobes and 214 microaerophilic organisms, 552 anaerobes and 154 microaerophilic isolates were identified up to species level giving concordant results by conventional techniques. Rest of the strains could be identified only up to genus level by biochemical characterization. MALDI-TOF MS was able to identify more isolates to the species level compared with conventional methods, including several new genera and species including Clostridia, Peptostreptococcus and Lactobacilli. Conclusion: Newly recognized and taxonomically rearranged genera, frequently isolated anaerobic and microaerophilic species as well as closely related species could be identified accurately by employing MALDI-TOF MS. Implementation of this technique for identification will shorten the Turn Around Time, reduce the cost in the Microbiology laboratory and improve the patient care with better clinical outcome. https://ijm.tums.ac.ir/index.php/ijm/article/view/5964 https://ijm.tums.ac.ir/index.php/ijm/article/download/5964/1907