Abstract <p>A series of three fluorobenzylidene-1,2,4-triazol-3-one derivatives―(<i>E</i>)-4-[(2-fluorobenzylidene)amino]- (<b>1</b>), (<i>E</i>)-4-[(3-fluorobenzylidene)amino]- (<b>2</b>), and (<i>E</i>)-4-[(4-fluorobenzylidene)amino]-5-methyl-2,4-dihydro-3<i>H</i>-1,2,4-triazol-3-one (<b>3</b>)―were synthesized and characterized by FTIR and ¹H and ¹³C NMR spectroscopy. Of the three synthesized isomeric (<i>E</i>)-4-(fluorobenzylideneamino)-5-methyl-2,4-dihydro-3<i>H</i>-1,2,4-triazol-3-one derivatives, isomers 1 and 2 are novel compounds, and compound 3 is previously known. A theoretical study was performed using the DFT/B3LYP/6–311++G(d,p) method. The molecular structures of compounds 1–3 were optimized, and their structural parameters were determined. Experimental FT-IR and NMR data were compared with calculated values, which confirmed the molecular structures and supported the experimental findings. The antibacterial and leishmaniacidal activities of compounds <b>1</b>–<b>3</b> were evaluated by the microdilution assay. <i>Streptococcus pneumoniae</i> was the most susceptible bacterium, while compound 2 was less effective against the tested bacteria than the other derivatives. Molecular docking analysis identified key molecular interactions responsible for the antileishmanial activity of compound <b>1,</b> demonstrating a high binding affinity for <i>Trypanothione reductase</i> (TRe).</p>