Varying transfection efficiencies and cytotoxicity are crucial aspects in cell manipulation. The utilization of gold nanoparticles (AuNP) has lately attracted special interest to enhance transfection efficiency. Conventional AuNP are usually generated by chemical reactions or gas pyrolysis requiring often cell-toxic stabilizers or coatings to conserve their characteristics. Alternatively, stabilizer- and coating-free, highly pure, colloidal AuNP can be generated by pulsed laser ablation in liquids (PLAL). Mammalian cells were transfected efficiently by addition of PLAL-AuNP, but data systematically evaluating the cell-toxic potential are lacking. Herein, the transfection efficiency and cytotoxicity of PLAL AuNP was evaluated by transfection of a mammalian cell line with a recombinant HMGB1/GFP DNA expression vector. Different methods were compared using two sizes of PLAL-AuNP, commercialized AuNP, two magnetic NP-based protocols and a conventional transfection reagent (FuGENE HD; FHD). PLAL-AuNP were generated using a Spitfire Pro femtosecond laser system delivering 120 fs laser pulses at a wavelength of 800 nm focusing the fs-laser beam on a 99.99% pure gold target placed in ddH2O. Transfection efficiencies were analyzed after 24h using fluorescence microscopy and flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of necrotic, propidium iodide positive cells (PI %). The addition of PLAL-AuNP significantly enhanced transfection efficiencies (FHD: 31 %; PLAL-AuNP size-1: 46 %; size-2: 50 %) with increased PI% but no reduced cell proliferation. Commercial AuNP-transfection showed significantly lower efficiency (23 %), slightly increased PI % and reduced cell proliferation. Magnetic NP based methods were less effective but showing also lowest cytotoxicity. In conclusion, addition of PLAL-AuNP provides a novel tool for transfection efficiency enhancement with acceptable cytotoxic side-effects.