The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of Gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial infections, including septic arthritis. However, the development of potent Sortase A inhibitors remains an unmet challenge. Sortase A relies on a five amino acid sorting signal (LPXTG), by which it recognizes its natural target. We report the synthesis of a series of peptidomimetic inhibitors of Sortase A based on the sorting signal, supported by computational binding analysis. By employing a FRET-compatible substrate, our inhibitors were assayedin vitro. Among our panel, we identified several promising inhibitors with IC50values below 200 μM, with our strongest inhibitor – LPRDSar – having an IC50of 18.9 μM. Furthermore, it was discovered that three of our compounds show an effect on growth and biofilm inhibition of pathogenicStaphylococcus aureus, with the inclusion of a phenyl ring seemingly key to this effect. The most promising compound in our panel, BzLPRDSar, could inhibit biofilm formation at concentrations as low as 32 μg mL−1, manifesting it as a potential future drug lead. This could lead to treatments for MRSA infections in clinics and diseases such as septic arthritis, which has been directly linked with SrtA.