Evidence over ability of Escherichia coli and Staphylococcus aureus to cause acute septic arthritis (ASA), and antibacterial potential of Paeonia lactiflora plant motivated present study to compare the inhibitory potential of hydroxyacetophenone derivative (HAD) and Paeonia lactiflora leaves extract (PLLE) against ASA triggering bacteria (ATB). Current study involved synthesis of HAD and preparation of PLLE. The HAD was characterized using ATR-IR, 1H-NMR and Mass spectrometric data. Both HAD and PLLE were further investigated for their antibacterial activity against ATB namely: Escherichia coli and Staphylococcus aureus. Among two, the HAD exhibited high antibacterial activity when compared with PLLE. Based on the results, present study concludes that HAD possess high antimicrobial potential against ATB and recommends that HAD should be further investigated to support its clinical significance.