Mastitis caused by multidrug-resistant Staphylococcus aureus and its robust biofilm formation poses a significant threat to dairy health. To address this, we developed a novel, eco-friendly therapeutic platform, Carthamus tinctorius L. extract-functionalized lignin nanoparticles (CTLe@LigNPs), using a one-pot ultrasound-assisted protocol. These nanoparticles demonstrated high colloidal stability (71.07 nm, ζ-potential of −21.7 mV), efficient drug loading, and sustained release in both gastric (~ 90%) and intestinal (~ 76%) environments over 24 h. The CTLe@LigNPs showed low cytotoxicity while effectively eradicating planktonic S. aureus (MIC = 125 µg/mL) and significantly disrupting pre-formed biofilms, achieving 71.38% inhibition at the highest concentration tested. In an oral gavage rat mastitis model, a single dose (100 mg/kg) for 7 days ameliorated mastitis symptoms and improved hematological parameters. Mechanistically, CTLe@LigNPs acted as a potent immunomodulator by downregulating TLR2/4, JNK, and MAPK signaling pathways, suppressing pro-inflammatory cytokines such as TNF-α and IL-1β by up to 50%, and elevating antioxidant enzymes (SOD, CAT, GPx) by over 30%. These findings establish CTLe@LigNPs as a promising multifunctional nano-antimicrobial and immunomodulatory platform for treating S. aureus mastitis, warranting further clinical translation.