Different bacterial species have developed resistance to antibiotics over the years. Penicillin analogs have been developed to counter these bacteria and in this study, we investigate the antibacterial efficacy of penicillin G and its analogs, amoxicillin, carbenicillin, piperacillin, cloxacillin, and ampicillin, against four strains of bacteria. Penicillin antibiotics mimic the D-Ala-D-Ala active site region of penicillin-binding proteins (PBP). PBPs are transpeptidases used in the synthesis of bacterial cell walls. Through a Kirby-Bauer assay, it was found that the inhibition performance of these penicillin-type antibiotics varies greatly among different bacterial strains. Moreover, to investigate the structural basis for the in vivo SAR observed, we also conducted in silico virtual screening via homology modeling, molecular docking, and density functional theory (DFT) calculations. We concluded that all six penicillin-type antibiotics inhibited S. epidermidis, E. coli, and N. sicca with varying degrees of efficacy but exhibited no inhibition against B. cereus. Our computational results suggested that the distance between the carbonyl-carbon of the beta-lactam ring is more influential on the antibacterial efficacy of the compounds than the thermodynamic binding affinity of these antibiotics to interact with the serine nucleophile. Penicillin G had the greatest broad-spectrum antibacterial activity with a high radius of inhibition against S. epidermidis, E. coli, and N. sicca.