Aims: Antibacterial effects of hydrogen peroxide and silver composition on selected pathogenic enterobacteriaceae was investigated in this study. Materials and Methods: The efficacy of 30 ppb silver in 0.3% hydrogen peroxide solution for inactivation of selected Enterobacteriaceae, including Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae was assessed for 72 hours in a designated nutrient broth medium and steel surface. The bactericidal growth ability was determined for each bacterium genus by the conventional colony count method and turbidimetry via an optical density (OD) assay at 450 nm in a time interval of 24 hours. Results: Suspensions of K.pneumoniae, and P.mirabilis showed a significant OD reduction at three 24-hour intervals (CI = 0.95; P < 0.05, for both), along with blocked growth in a designated broth medium during 24 to 48 hours of exposure. The disinfectant was also significantly efficient for inactivating of the mentioned bacteria on steel surfaces after a 15-minute time exposure (CI = 0.95; P < 0.05). For E.coli, the OD decreased slightly during the initial exposure time, but increased after 24 hours. Viable E.coli cells were proved by colonies grown on the plate. A qualitative surface decontamination test showed that three pathogenic bacteria were inactivated significantly after disinfectant exposure (CI = 0.95, P < 0.05). Conclusions: In conclusion, a combination of hydrogen peroxide and silver ions was proposed as a strong disinfecting agent both in suspensions and on the surfaces against these three important human pathogens.