In present study, nineteen novel trimethoprim (TMP) derivatives were designed, synthesized and evaluated for their antibacterial potential. Hydroxy trimethoprim 2 (HTMP) was synthesized by following the demethylation of 4-methoxy group at trimethoxy benzyl ring of TMP. Structure–activity relationship (SAR) studies were explored on HTMP by incorporating various substituents leading to the identification of some new compounds with improved antibacterial activities. The results revealed that the introduction of benzyloxy (4a–e) and phenyl ethanone (5a–e) group at 4-position of dimethoxy benzyl ring leads to overall increase in the antibacterial activity. The most potent antibacterial compound discovered is benzyloxy derivative 4b with MIC value of 5.0 μM against Staphylococcus aureus and 4.0 μM against Escherichia coli strains higher than the standard TMP (22.7 μM against S. aureus and 55.1 μM against E. coli). Substitution at 4-NH₂ group was not tolerated and the resulting Schiff base derivatives 3a–h demonstrated very little or no antibacterial activity in the tested concentration domain. We further performed exploratory docking studies on dihydrofolate reductase (DHFR) to rationalize the in vitro biological data and to demonstrate the mechanism of antibacterial activity. For the ability to cross lipophilic outer membrane, log P was computed. It was found that the compounds possessing high hydrophobicity have high activity against E. coli.