Nanotechnology provides solutions by combining the fields of textiles and medicine to prevent infectious microbial spread. Our study aimed to evaluate the antimicrobial activity of nanofiber sheets incorporated with a well-known antibiotic, ceftriaxone. It is a third-generation antibiotic that belongs to the cephalosporin group. Different percentages (0, 5%, 10%, 15%, and 20%; based on polymer wt%) of ceftriaxone were incorporated with a polymer such as polyvinyl alcohol (PVA) via electrospinning to fabricate nanofiber sheets. The Kirby-Bauer method was used to evaluate the antimicrobial susceptibility of the nanofiber sheets using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). For the characterization of the nanofiber sheets incorporated with the drug, several techniques were used, such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). Our results showed that the nanofiber sheets containing ceftriaxone had potential inhibitory activity against E. coli and S. aureus as they had inhibition zones of approximately 20–25 mm on Mueller-Hinton-agar-containing plates. In conclusion, our nanofiber sheets fabricated with ceftriaxone have potential inhibitory effects against bacteria and can be used as a dressing to treat wounds in hospitals and for other biomedical applications.