The bacterial cell wall and enzymes involved in peptidoglycan biosynthesis are prime targets for the discovery of novel antibacterial agents. Among these enzymes, d-alanine-d-alanine ligases (Ddl) are particularly significant due to their utilization of specific substrates (d-amino acids) essential for bacterial viability. Isozymes of Ddl that utilize alternative substrates such as d-lactate or d-serine are found in vancomycin-resistant Gram-positive bacteria, initially identified in Enterococcus species, and now represent a growing concern in clinical settings. In this study, we isolated and identified vancomycin-resistant Enterococcus casseliflavus (E. casseliflavus) strain SSK and used it for amplification, cloning, and purification of the vanC2 type of d-alanine-d-serine ligase (EcfDdls). Investigations of substrate specificity and enzyme kinetics provided insights into the enzyme's mechanistic action. Evaluation of the inhibitory potential of the previously virtually screened oxadiazole derivative 1-[(5-methyl-1,2-oxazol-3-yl)methyl]-4-{[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]methyl}piperazine (CID 45805715) was carried out using an inorganic phosphate detection assay, which demonstrated complete enzymatic inhibition of purified EcfDdls. When tested, CID 45805715 significantly inhibited activity of Ddl, with an IC(50) of 76.7 μM, compared to 313 μM for the reference compound DCS. Moreover, this compound also exhibited antimicrobial activity against vancomycin-resistant E. casseliflavus strain SSK. Thus, these findings provide valuable insights into the activity and inhibition of vanC2 EcfDdls, offering a promising avenue for addressing vancomycin resistance in enterococci, particularly in nosocomial infections affecting immunocompromised patients.