Wound infection is a serious threat to patients, in particular those with septic wound infections, which result in high mortality rates. Moreover, the treatment of wound infections with antimicrobial-resistant and/or biofilm-forming pathogens can be challenging. Nisin, a potent antimicrobial against Gram-positive bacterial pathogens, has been used in the food industry as a preservative for decades. Silver has been approved by the FDA as a topical antimicrobial. Here, we show that silver-nisin nanoparticles (Ag-nisin NP), with an average diameter of 60 nm, can be quickly synthesized with the assistance of a simple microwave. Ag-nisin NP act as bactericidal antibiotics against the tested pathogens. In contrast, resistance was observed in S. aureus and A. baumannii that were treated with silver nitrate alone. In addition, Ag-nisin NP showed potent antibiofilm activity against S. aureus, P. aeruginosa, A. baumannii, K. pneumoniae, and E. coli, which are pathogens occurring in wound infections. Notably, the synthesized Ag-nisin NP showed lower cytotoxicity than silver nitrate to human cells. This formulation provides an alternative and safe measurement for biofilm-infected wound control.