Bovine mastitis is one important metabolic disease in dairy cows caused by S. aureus and E. coli impacting huge economic losses worldwide. Silver Nanoparticles (AgNPs) possess unique electrical, thermal, optical, and biological properties, scrutinizing them as more effective antimicrobial agents. This study aimed to synthesize and evaluate AgNPs as effective antimicrobial compounds against S. aureus and E. coli. In the present work, the extract of citrus limon was analysed with gas chromatography-mass spectrometry (GC-MS) analysis, citric acid was found abundant in methanolic solvent. Synthesis of AgNPs was made by both green and chemical synthesis method, charctererized by UV‒visible spectroscopy, electron microscopy, zeta sizing and infrared spectroscopy. The result confirmed that both green and chemical AgNPs are in nanoscale range with average size ranged from 10 to 20 nm. The zeta potential was found negative and green synthesized AgNPs resulted significantly (P ≤ 0.05) higher antimicrobial activities than chemical synthesized AgNPs and also commercial available antibiotics. The minimum inhibitory concentration (MIC) was also evaluated and green synthesized AgNPs resulted MIC