The inhibition or prevention of biofilm formation represents an emerging strategy in the war againstantibiotic resistance, interfering with key players in bacterial virulence. This approach includes the inhibitionof the catalytic activity of transpeptidase sortase A (Srt A), a membrane enzyme responsible forcovalently attaching a wide variety of adhesive matrix molecules to the peptidoglycan cell wall in Grampositivestrains. A new series of seventeen 1,2,4-oxadiazole derivatives was efficiently synthesized andscreened as potential new anti-virulence agents. The ability of inhibiting biofilm formationwas evaluatedagainst both Gram-positive and Gram-negative pathogens. Remarkably, all these compounds inhibitedS. aureus and/or P. aeruginosa biofilm formation in a dose dependent manner, with 50% biofilm inhibitoryconcentrations (BIC50s) below 10 mM for the most active compounds. Inhibition of SrtA was validated asone of the possible mechanisms of action of these new 1,2,4-oxadiazole derivatives, in the tested Grampositivepathogen, using a specific enzymatic assay for a recombinant S. aureus SrtA. The three mostactive compounds, eliciting BIC50 values for S. aureus ATCC 25923 between 0.7 and 9.7 mM, showed agood activity toward the enzyme eliciting IC50 values ranging from 2.2 to 10.4 mM.