Staphylococcus aureus(S. aureus) is a common bacterial cause of bovine mastitis known for its resistance to antimicrobials through various mechanisms includingdrug efflux pumping, which aids in its colonization in theudder tissue and spread in the environment. Thymoquinone (TQ) extracted from Nigella sativa(N. sativa), is recognized for its strong antibacterial properties against S.aureus.This study aimed to evaluate the effect of TQ on drug efflux pumping in S. aureus. Herein,we collected 67 milk samples from cows with chronic mastitis to isolate S. aureus, assess its resistance to antimicrobials, and to analyze the relationship between TQ's minimum inhibitory concentration (MIC) and its inhibition zone diameters (IZDs) using the disc diffusion, as well as ethidium bromide cartwheel (EtBr CW) and accumulation assays. Out of the 67 samples, 16 (23.9%) yielded S. aureus isolates, which exhibited high resistance to several antimicrobials, with the highest resistance (87.5%) against each of cefepime and azithromycin. There was no correlation between the MIC values of TQ and the IZD for discs containing 50 µg of TQ. The efflux assay demonstrated that TQ functions as an efflux pump inhibitor (EPI) similar to verapamil for some S. aureus isolates.The study concluded that the antibacterial effectiveness of TQ is largely due to its role in inhibiting drug efflux pumps, a mechanism prevalent among the tested isolates and comparable to that of verapamil. Additionally, MIC detection could not reliably predict the results from disc diffusion testing.