[Objective] The objective of the study was to assess the antimicrobial activity of silver nanoparticles (AgNPs) against multiple drug resistant strains. [Methods] Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AgNPs against three model microbes, namely Escherichia coli, Staphylococcus aureus, Candida albicans were measured by microdilution broth method. Time-kill curve within 24 h was made according to colony count method after three model microbes were treated with a series concentration of AgNPs. Post-antibiotic effect was tested by colony count method. Finally, we determined the antimicrobial efficacy against multiple drug resistant strains in biological safety laboratory grade 2 (BSL-2). [Results] AgNPs with a diameter of 5 nm to 30 nm were synthesized by the biological method. The zeta potential was -19.5 mV. The time-kill curve of the three model microbes showed time-dependent antibacterial activity. The effect of AgNPs on E. coli and C. albicans after "antibiotic effect" increased with time, there was no obvious "post-antibiotic effect" on S. aureus. Both MIC values and MBC values of AgNPs for the three model microbes were between 1 μg/mL and 4 μg/mL. However, the MIC value of AgNPs for the three human multidrug-resistant strains was 6 μg/mL to 26 μg/mL and MBC value of AgNPs was 10 μg/mL to 32 μg/mL. The MIC values of AgNPs for 14 animal multi-drug resistant strains were between 4 μg/mL and 10 μg/mL, and the MBC values were between 8 μg/mL and 16 μg/mL. The MBC/MIC values of all the tested strains were less than 2. [Conclusion] AgNPs is a time-dependent antimicrobial agent with different "post-antibiotic effect", which can inhibit both human and animal-derived multi-drug resistant bacteria.