The most effective small molecular inhibitors of TrmD with confirmed in vivo antibacterial activity contain either 3-indolyl of thieno[2,3-d]pyrimidine moiety. Recently we have reported the antimicrobial activity of the synthetically available 4-methylthieno[2,3-d]pyrimidine-6-carboxamides with benzyl substituents at the primary amide fragment as effective antimicrobials with the predicted affinity to TrmD isolated from P. aeruginosa. It was also reported that 4-oxothieno[2,3-d]pyrimidine derivatives were more effective TrmD inhibitors rather than their 4-alkoxy analogues with aromatic pyrimidine fragment. Therefore we have prepared N-(benzyl)-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamides as the close analogues, which turned out to be active in vitro against the strains of S. aureus and B. subtilis and only moderately active against P. aeruginosa strain. Docking studies showed that despite the good values of the scoring functions, the conformational analysis of the ligands’ poses in the active site revealed their ability for only partial inhibition of TrmD of P. aeruginosa. The study revealed better activity for N-benzyl-4,5-dimethylthieno[2,3-d]pyrimidine-6-carboxamide against P. aeruginosa in comparison to their 4-oxo analogues.