Microbial resistance to the current available antibiotics is considered a global health problem, especially for the Multi-Drug Resistant pathogens (MDR) including methicillin resistant Staphylococcus aureus. Recently nanoparticles (NPs) have been involved in variety of antimicrobial applications due to their unique properties of antibacterial effects. However, the molecular mechanisms behind their antibacterial activity are still not fully understood. In this study, we produced silver Ag NPs (average size 27 nm) and silver-Titanium Ag-TiO2 NPs (average size 47 nm) using picosecond laser ablation. Our results showed that both laser NPs had obvious size-dependent antibacterial activity. The laser Ag NPs with a size of 19 nm and Ag-TiO2 NPs with a size 20 nm presented the highest bactericidal effect. The laser generated Ag and Ag-TiO2 NPs with concentrations 20, 30, 40, and 50 Î¼g/ml showed strong antibacterial effect against three bacterial strains: E. coli, P. aeruginosa, and S. aureus, and induced the generation of reactive oxygen species (ROS), lead to cell membrane interruption, lipid peroxidation, DNA damages, glutathione depletion and the eventual cell death. Both types of laser NPs at two concentrations (2.5 and 20 Î¼g/ml) showed low cytotoxicity to the in vitro cultured five types of human cells originated from the lung (A549), kidney (HEK293), Liver (HepG2), skin (HDFc) and blood vessel cells (hCAECs). The antibacterial activity of the laser generated Ag and Ag-TiO2 NPs had lasted for over one year depending on the degree of air exposure and storage conditions. Frequent air exposure increased particle oxidation and reduced the antibacterial durability of the laser generated Ag NPs. The laser generated Ag NPs had lower antibacterial activity when stored in cold compared to that stored at room temperature. The antibacterial activity of laser generated Ag and Ag-TiO2 NPs were also compared with four types of commercial based-silver wound dressings (Acticoat TM, AquacelÂ® Ag, Contreet Â®Foam, and UrgotulÂ® SSD) against E. coli to inform future application in this area. In conclusion, laser generated Ag and Ag-TiO2 NPs have strong bactericidal effect and low toxicity to human cells which could be a type of promising antibacterial agents for future hygiene and medical applications.