Background: The rapid rise of multidrug-resistant bacteria is undermining conventional antibiotics, driving the search for novel scaffolds. Pongamia pinnata, a leguminous medicinal plant rich in flavonoids, karanjin, and pongamol, represents an untapped source of antibacterial leads with strong translational potential.

Methods: Bioactive fractions from P. pinnata seeds and leaves were extracted using methanol and ethyl acetate, followed by LC-MS and NMR characterization. Antibacterial activity was assessed against MDR Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli via CLSI-standard broth microdilution. Biofilm formation and eradication assays were quantified by crystal violet staining and confocal microscopy. Synergy with ceftriaxone and azithromycin was tested using checkerboard and time–kill kinetics. BALB/c mice infected with MDR S. aureus were used for in vivo efficacy and toxicity evaluation.

Results: P. pinnata fractions showed strong antibacterial effects, with MICs of 16 µg/mL (S. aureus), 32 µg/mL (P. aeruginosa), and 24 µg/mL (E. coli). Biofilm inhibition exceeded 72% at sub-MIC levels, while eradication of mature biofilms reached 61% at 32 µg/mL. Synergistic assays revealed marked potentiation with ceftriaxone (FICI 0.34) and azithromycin (FICI 0.39), reducing effective antibiotic concentrations by fourfold. In vivo, alkaloid-rich fractions (50 mg/kg, i.p.) improved survival to 78% versus 18% in controls and reduced bacterial loads in spleen and liver by >2.5 log CFU. Histological analysis confirmed no acute toxicity in major organs.

Conclusion: Pongamia pinnata phytochemicals exhibit robust antibacterial, anti-biofilm, and synergistic activity with conventional antibiotics, validated by in vivo efficacy. These results position P. pinnata as a powerful natural reservoir for developing next-generation antibacterial leads against MDR pathogens.