Background. DNA adenine methyltransferase (dam) has been well documented for its role in regulation of replication, mismatch repair and transposition. Recent studies have also suggested a role for dam in protection against antibiotic stress, although this is not yet fully defined. We therefore evaluated the role of dam in the development of antibiotic resistance and triclosan-associated cross-resistance. Results. A significant impact on growth rate was seen in the dam knockout compared to the parental strain. Known triclosan resistance-associated mutations in fabI were seen regardless of dam status, with an additional mutation in lrhA seen in the dam knockout. The expression of multiple antibiotic resistance-associated genes was significantly different between the parent and dam knockout post-resistance induction. Reversion rate assays showed that resistance mechanisms were stable. Conclusions. dam knockout had a significant effect on growth, but its role in the development of antibiotic resistance is likely confined to those antibiotics using acrAD-containing efflux pumps.