DNA methylation is an important epigenetic regulator of gene expression. Abnormalities in DNA methylation patterns have been associated with various developmental and proliferative diseases, particularly cancer. Targeting DNA methyltransferases (DNMTs) represents a promising strategy for the treatment of such diseases. Current DNMT inhibitors suffer important drawbacks with respect to their efficacy, specificity, and toxicity. In this study, we have set up a robust in vitro bacterial M.SssI DNMT activity assay to systematically screen a collection of 26 240 compounds that were predicted to compete with the S-adenosyl-L-methionine (SAM) substrate of DNMT. This resulted in the identification of a novel set of structurally distinct inhibitors of M.SssI DNMT activity. Although molecular docking studies using an M.SssI homology model suggest that these compounds might compete with SAM binding, mode of activity (MoA) assays are still needed to confirm this hypothesis. Our set of novel M.SssI DNMT inhibitors, once confirmed in an orthogonal DNMT assay, may thus serve as a starting point to identify and characterize suitable lead candidates for further drug optimization.