Methicillin-resistant Staphylococcus epidermidis (MRSE) is an emerging multidrug-resistant pathogen responsible for numerous healthcare-associated infections. Most of them are resistant to all classes of antibiotics and thus lead to therapeutic impasse. For this reason, identifying new targets and characterizing new drugs are essential. We recently showed that methicillin-resistant Staphylococcus aureus strains deficient in D-alanylation of teichoic acids (TAs) lost resistance to various β-lactams. Here we explore if D-alanylation of TAs might be a druggable target to overcome β-lactam resistance of MRSE using a competitive DltA inhibitor. The binding affinity of a DltA inhibitor with the purified DltA protein was monitored by determining the half maximal inhibitory concentration (IC50). The efficiency of D-alanylation inhibition was determined by quantifying the ester-linked D-alanine content of purified TAs. Minimal inhibition concentrations (MICs) and bactericidal effects of several β-lactams were monitored in the absence or presence of the inhibitor against a panel of clinical MRSE isolates. Finally, the ability of inhibition of D-alanylation (i) to rescue MRSE-infected larvae of Galleria mellonella and (ii) to prevent or eradicate S. epidermidis biofilms was evaluated. The DltA inhibitor showed IC50 in the low µM range, drastically reduced the D-alanine esters content of TAs and re-sensitized MRSE to β-lactams. The most effective treatment was the DltA inhibitor/imipenem combination. Finally, inhibition of D-alanylation significantly reduced the virulence of MRSE in the G. mellonella infection model and strongly reduced the ability of S. epidermidis to form biofilms. All together, our results show the promising nature of the D-alanylation of TAs as a therapeutic target to fight against MRSE infections.