In the current investigation, silver/gold nanoparticles (NPs) were synthesized using two methods: chemical and biological, and then characterized colloidal solutions of both NPs using UV-Vis, transmission electron microscopy (TEM) and zeta potential analyzers, X-ray powder diffraction (XRD), and energy dispersive X-ray (EDX) as well as the ToxTrak test for in vitro toxicity and antibacterial activity against Gram-positive bacteria (B. subtilis) and Gram-negative bacteria (E. coli). The plasmon peak of chemical synthesized silver NPs (CH-AgNPs) and gold NPs (CH-AuNPs) was observed at 414 and 530 nm, respectively, while the sharp plasmon peak of biological synthesized silver NPs (Bio-AgNPs) and gold NPs (Bio-AuNPs) was observed at 410 and 525 nm. Under transmission electron microscopy (TEM), the average sizes of CH-AgNPs and CH-AuNPs were 50.56 and 25.98 nm, respectively. Bio-AgNPs and Bio-AuNPs, on the other hand, had average sizes of 25.25 and 16.65 nm, respectively. The stability of NPs was also investigated using the zeta potential. The crystalline structure of AgNPs was confirmed through XRD, and EDX results confirm the element compositions. In the ToxTrak test, the toxic effect value/percentage inhibition (TEV/PI) was calculated. The results showed that CH-AgNPs have the highest TEV/PI value (85.45% for B. subtilis and 83.77% for E. coli) when compared to Bio-AgNPs (55.75% for B. subtilis and 54.42% for E. coli). CH-AuNPs, on the other hand, were 33.51% toxic to B. subtilis and 36.85% toxic to E. coli, compared to Bio-AuNPs, which were 23.36% toxic to B. subtilis and 24.46% toxic to E. coli. The antibacterial activity of Ag/Au NPs was tested and monitored; zone of inhibition (mm in diameter) against B. subtilis and E. coli, with the following pattern emerging: CH-AgNPs (24.80) had the highest antibacterial activity followed by Bio-AgNPs (22.80) < CH-AuNPs (10.60) < Bio-AuNPs (09.00), whereas the control sample (tetracycline antibiotic) revealed a 25.08 mm, zone of inhabitation. Overall, Bio-AgNPs and Bio-AuNPs are the most effective pathogen-killing materials with the lowest toxicity. Our suggestion is that such materials instead of chemical synthesized NPs can be used to coat antibiotic drugs and could be a game-changer for the pharmaceutical industry in terms of effectively controlling the pathogenic bacteria.