MALDI-TOF MS has revolutionized the identification of microorganisms and has become an indispensable part of routine diagnostics in the clinical microbiological laboratory. However, application of this technique in microbial surveillance outside of clinical settings is limited. In this study, we have evaluated the performance of a Bruker MALDI Biotyper System for the identification of bacteria isolated from the hand palms of fresh produce handlers and their surrounding environments in a wholesale fresh produce market in Doha, Qatar. The accuracy was verified against the results obtained by bacterial 16S rRNA gene sequencing. A total of 105 isolates were tested, of which 67 (64%) isolates were identified by MALDI-TOF MS and 101 isolates (96%) were identified by 16S rRNA gene sequencing, either at the genus level or species level. However, MALDI-TOF MS identified more isolates (41%) at the species level than 16S rRNA gene sequencing (28%). MALDI-TOF MS was particularly useful in the species level identification of Enterobacteriaceae. MALDI-TOF MS successfully identified most known human pathogens in a rapid and cost-effective manner but failed to identify a significant number of isolates that were of environmental origin, suggesting room for further expansion of the reference database.