The spermicidal effects of silver nanoparticles (AgNPs) hinder its application in the field of artificial insemination. In this study, silver-carbon NPs (Ag@C NPs) was synthesized and applied as an alternative antibiotic agent for bull semen extender. Ag@C NPs were characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), atomic absorption flame spectroscopy, transmission electron microscope (TEM), and high-resolution TEM (HR-TEM). Data analysis revealed the successful synthesis of Ag@C NPs with a particle size of 1-5 nm (average particle size of 2.5 nm) embedded into carbon. The antimicrobial activity of Ag@C NPs was tested against bacteriospermia of fresh semen collected from five fertile bulls (three ejaculates/bull). Escherichia coli (E. Coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) were isolated from fresh semen samples and identified by culture, staining, and conventional biochemical tests. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ag@C NPs against bacteriospermia was determined at 5 and 37 ºC. Ag@C NPs showed efficient antimicrobial activity (MIC: 3.125 - 12.5 μg/mL) against the tested strains and strong bactericidal effect on S. aureus, and P. aeruginosa (MBC: 3.125 μg/mL), with no detrimental effect (P ˃ 0.05) on the percentage of sperm motility (70.71±4.82; 74.65±4.46), plasma membrane integrity (68.39±4.31; 72.38±4.91), acrosome integrity (88.40±13.21; 86.77±14.23), and normal sperm morphology (86.85±7.43; 87.82±8.15) at concentrations of 15 and 30 μg/mL, respectively, after a cold storage of 48 h. However, Ag@C NPs showed a detrimental effect on sperm parameters in a dose dependent manner at concentrations ≥60 μg/mL. Ag@C NPs showed no adverse effect on the sperm´s ultrastructure with limited sperm internalization at MIC. In conclusion, Ag@C NPs could be used as an alternative antibiotic agent for bull semen extender without a significant cytotoxic effect on the sperm during cold storage. However, further investigations for their effects on embryo production and female genitalia are still required.