Several recent biological science studies have been focused on nanotechnology and nanomaterials due to their potential use in biomedicine. Drug delivery systems are an example of biomedical applications utilizing nanoparticles. Silver nanoparticles (AgNPs) can be used for these drug delivery systems. However, the effects of cytotoxicity caused by AgNPs are not fully understood. Determining the optimal characteristics to facilitate the biocompatibility of AgNPs is an important subject for application. In the present study, human erythrocytes were used as an in vitro model to examine the size, dose, and coating surfactant-dependent cytotoxicity of AgNPs. Our results demonstrated that polyvinylpyrrolidone (PVP) was a more suitable surfactant than polyethylene glycol (PEG) for AgNPs capping. In addition, we determined the appropriate particular size and dosage of AgNPs to reduce human erythrocytes hemolysis. Membrane damages including hemolysis, potassium efflux, protein leakage, and alterations in cell shape and membrane fragility were minimized with 100-nm AgNP particles. This study provides novel insights into AgNPs cytotoxicity and a basis for utilizing AgNPs for diagnostic and therapeutic applications.