The emergence of antibiotic-resistant pathogens has led to a growing demand for novel and effective antimicrobial agents. In our study, a novel 3-(2-chloroacetyl) oxazolidine-2-one was synthesized and screened for its antimicrobial activity against thirteen pathogenic bacteria and five fungal species. The results showed that the new compound, L1, exhibited potent antibacterial activity, with inhibition zones ranging from 14 to 25 mm and minimum inhibitory concentrations (MICs) of 125 to 500 µg/mL. Regarding antifungal activity, all strains were susceptible to the tested molecule, with inhibition zone diameters ranging from 16 to 36.5 mm and MICs ranging from 7.81 µg/mL to 250 µg/mL, Tests against virulence factors demonstrated that the new molecule significantly inhibited biofilm formation, motility and quorum sensing. In terms of antioxidant capacity, the molecule tested showed a (2, 2-diphenyl-1 picrylhydrazyl) (DPPH) scavenging activity with an IC50 value of 5.58 ± 0.02 μg/mL, and an anti-inflammatory effect demonstrated a percentage inhibition of about 84.38 ± 5.11%. For the HOMO-LUMO analysis, the compound has a positive log p-value, confirming its lipophilicity and biological activity. A molecular anchoring study was performed to understand their binding modes in the active site of the Staphylococcus aureus Thymidylate Kinase (TMK) binding site. In conclusion, these results are promising for the novel 3-(2-chloroacetyl) oxazolidine-2 in medical and pharmaceutical applications.