Increased resistance to anti-infective drugs, in particular multidrug resistance Staphylococcus aureus (MRSA), is a global concern to health. Aminoacyl-tRNA synthetase enzymes are potential targets for the development of new antibiotics for MRSA infections. Phenylalanine tRNA synthetase enzyme (PheRS) was selected because it is structurally unique (tetrameric (αβ)2) among the aaRS enzymes and considerably different at the amino acid sequence level than human cytosolic and human mitochondrial aaRS. There is no crystal structure available for S. aureus PheRS, therefore comparative structure modeling is required to establish a putative 3D structure for the required enzyme enabling development of new inhibitors. The S. aureus PheRS alpha subunit homology model was constructed using Molecular Operating Environment (MOE) software. Staphylococcus haemolyticus PheRS was the main template while Thermus thermophilus PheRS was utilised to predict the enzyme binding with tRNAphe. The model has been evaluated and the query protein active site was predicted from its sequence using a conservation analysis tool. Docking suitable ligands using MOE into the constructed model was used to assess the predicted active sites. The docked ligands involved the PheRS natural substrate (phenylalanine), phenylalanyl-adenylate and several described S. aureus PheRS inhibitors. Molecular modelling and docking studies were performed to explore the possibility of the proposed novel compounds to bind with the target enzyme using MOE and LeadIT software. Series I and II were synthesised depending on the natural substrate L-phenylalanine. However, the compounds did not show activity against the S. aureus (MIC ≥ 128 mg/mL). Series III to XII were developed to include either adenine or a biaryl mimic (benzimidazole or indole) to represent the adenyl moiety of phenylalanyl adenylate. The ‘adenyl’ portion was linked, through a 3-4 atoms linker that spans the hydrophobic channel, to a heterocyclic 5-membered ring having either thiol or nitrogen or both to make a H-bond with the key binding amino acid residue acidic Glu216, and finally the remainder of the compound, which may be aliphatic or aromatic, to fill the large hydrophobic pocket and may contributed with H-bond interactions. Although the compounds did not show activity against the S. aureus (MIC ≥ 128 μg/mL), screening of the compounds against a broad microbial panel showed activity against vanA and vanB resistant strains of Enterococcus faecalis represented in compounds 7e, 12n, 23a: 23h, 24b, 24c, 29a, 29b, 34a and 44b. The 1,3,4-oxadiazole 23f, activity was also observed against Staphylococcus aureus sensitive and mecA resistant strains (32 μg/mL). The synthesised compounds were microbiologically tested for P. aeruginosa PheRS and S. pneumonia PheRS inhibition through aminoacylation assay due to the availability of these enzymes and their high degree of similarity with S. aureus PheRS.