Aims The present study aimed to investigate the anti-Staphylococcus aureus and anti-biofilm properties of 4-methoxy-1-methyl-2-oxopyridine-3-carbamide (MMOXC) on S. aureus UDP-MurNAc-pentapeptide (MurF), peptidyl deformylase (PDF) and uridine monophosphate kinase (UMPK). Methods and Results The invitro efficacy of MMOXC was evaluated using quantitative polymerase chain reaction, invitro assays and broth microdilution methods. Further, the minimum inhibitory concentration (MIC), IC50 and zone of inhibition were recorded in addition to the anti-biofilm property. MMOXC inhibited pure recombinant UMPK and PDF enzymes with a K-i of 0.37 and 0.49 mu moll(-1). However K-i was altered for MurF with varying substrates. The MurF K-i for UMT, (D)-Ala-(D)-Ala and ATP as substrates was 0.3, 0.25 and 1.4 mu moll(-1), respectively. Real-time PCR analysis showed a significant reduction in PDF and MurF expression which correlated with the MIC90 at 100 mu moll(-1) and IC50 in the range 421 +/- to 1.5 to 50 +/- 1 mu moll(-1) against all strains tested. At 5 mu moll(-1) MMOXC was able completely to remove preformed biofilms of S. aureus and other drug resistant strains. Conclusions MMOXC was able to kill S. aureus and drug resistant strains tested by inhibiting MurF, UMPK and PDF enzymes and completely obliterated preformed biofilms. Significance and Impact of the Study Growth reduction and biofilm removal are prerequisites for controlling S. aureus infections. In this study MMOXC exhibited prominent anti-S. aureus and anti-biofilm properties by blocking cell wall formation, RNA biosynthesis and protein maturation.