Antimicrobial resistance is a significant public health problem, and the use of nanotechnology to control bacterial growth has increased, including multidrug-resistant ones. In this context, nanoparticles of different elements, mainly metals, such as silver, gold, and iron, have been extensively researched due to their efficient antimicrobial activity. Another element that stands out is selenium, which nanoparticles have high bioactivity and antibacterial action, although few studies report its use as a metallic nanoparticle. Therefore, the objective of this study was to perform the synthesis of selenium nanoparticles (SeNPs) from extracts of three plants: Allium cepa (onion), Malpighia emarginata (acerola), and Gymnanthemum amygdalinum (boldo). The synthesized nanoparticles were characterized by Dynamic Light Scattering (DLS), scanning electron microscopy (SEM), Fourier-Transform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (DRX). Antibacterial activity was evaluated by microdilution in broth, followed by the time-kill curve analysis. The bacterial strains tested were Streptococcus agalactiae, Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and Escherichia coli. Hemolytic activity was also determined. The synthesized SeNPs had a size between 245 and 321 nm and mostly spherical morphology. Antimicrobial activity against all Gram-positive bacteria tested was observed, with minimal inhibitory concentrations ranging from 6.125 to 98 μg/ml, but without action in the tested Gram-negative bacteria. Low hemolytic capacity was observed, with CC50 ranging from 0.82 to 2.1 mg/ml. The antimicrobial activity and low hemolytic concentration indicate the possibility of use against Gram-positive bacteria, including multidrug-resistant ones, opening a wide variety of options for their applications.