Mastitis is a significant inflammatory condition of the mammary gland in dairy cows. It is caused by bacterial infections and leads to substantial economic losses worldwide. The disease can be either clinical or sub-clinical and presents challenges such as reduced milk yield, increased treatment costs, and the need to cull affected cows. The pathogenic mechanisms of mastitis involve the activation of Toll-like receptors (TLRs), specifically TLR2 and TLR4. These receptors play crucial roles in recognizing pathogen-associated molecular patterns (PAMPs) and initiating immune responses through the NF-κB signaling pathway. Recent in vitro studies have emphasized the importance of the TLR2/TLR4/NF-κB signaling pathway in the development of mastitis, suggesting its potential as a therapeutic target. This review summarizes recent research on the role of the TLR2/TLR4/NF-κB signaling pathway in mastitis. It focuses on how the activation of TLRs leads to the production of proinflammatory cytokines, which, in turn, exacerbate the inflammatory response by activating the NF-κB signaling pathway in mammary gland tissues. Additionally, the review discusses various bioactive compounds and probiotics that have been identified as potential therapeutic agents for preventing and treating mastitis by targeting TLR2/TLR4/NF-κB signaling pathway. Overall, this review highlights the significance of targeting the TLR2/TLR4/NF-κB signaling pathway to develop effective therapeutic strategies against mastitis, which can enhance dairy cow health and reduce economic losses in the dairy industry.