This study aimed to assess antibacterial activity of Knema retusa wood extract (KRe) against antibiotic resistant staphylococci which are causative agents of bovine mastitis. From 75 cases of intramammary infections in dairy cows, 66 staphylococcal isolates were collected, including 11 Staphylococcus aureus isolates (17%) and 55 coagulase-negative staphylococci (83%). Sixty isolates (91%) formed strong biofilms. KRe had minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against the isolates ranging 32-256 ug/mL and 64-512 ug/mL, respectively. Two-hour KRe exposures at 4×MIC, viabilities of S. aureus and S. haemolyticus decreased by 3 log10 compared to the control. Scanning EM (SEM) showed that KRe disrupted the bacterial cells of both species. KRe at 1/16×MIC significantly inhibited biofilm formation (P