Objectives: To evaluate the activity of Methylglyoxal against the blood culture isolates of Salmonella Typhi and various Gram negative rods and to compare the activity of Methylglyoxal against S. Typhi and other Gram negative rods. Methods: It was an experimental study conducted at the Department of Microbiology, University of Health Sciences (UHS), Lahore-Pakistan in collaboration with the Department of Microbiology, CMH Lahore, from July 2011 to June 2012. Recent blood culture isolates of S. Typhi and other Gram negative rods were collected from different hospitals of Lahore and kept stored at -80°C. As per the latest CLSI guidelines, morphological, biochemical and serological identification was carried out and antimicrobial susceptibility was tested. A multi-point inoculator was used to carry out agar dilution for determination of MICs of MGO. Results were determined after compilation of data using latest SPSS version. Results: MIC90 of MGO against the clinical isolates of S. Typhi was 0.20 mg/mL (2.8 mM) and against Gram negative rods it was 0.21 mg/mL (3.0 mM). The p-value of MICs of MGO against the isolates of S. Typhi was 0.023 when compared with Gram negative rods (p<0.05; statistically significant). Conclusion: MGO has a scientifically proven in vitro antimicrobial activity against blood culture isolates of S. Typhi and various Gram negative rods. doi: https://doi.org/10.12669/pjms.35.4.807 How to cite this:Afzal RK, Khalid F, Hannan A, Ahmed SA. Methylglyoxal: Antimicrobial activity against blood culture isolates of Salmonella Typhi and other Gram negative rods. Pak J Med Sci. 2019;35(4):1110-1114. doi: https://doi.org/10.12669/pjms.35.4.807 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.