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Carbon nanomaterials are ground-breaking two-dimensional materials with a wide range of applications because of their unique properties, which include excellent optical, electrical, thermal, and mechanical capabilities; biocompatibility; and inexpensive large-scale production costs. In this study, carbon nanostructural materials (CNMs) were effectively generated using a pulsed laser ablation method on a graphite target immersed in deionized water, and their cytotoxicity and antibacterial activities were evaluated. Lasing pulse duration, ns (1064 nm) with different laser energies (60-220 mJ), was employed for irradiating the target. The formation of CNMs was analyzed using Fourier transform infrared spectroscopy, which demonstrated that C–H, C–C, C–O, and C=C bonds were successfully formed. TEM micrographs observed CNM formation with a spherical shape and size ranging from 20 to 90 nm. The absorbance was measured using UV-Vis spectroscopy; it increased with increasing laser energy showing two broad shoulders around 210 and 305 nm. Thereafter, the antibacterial activity was tested against Escherichia coli and Staphylococcus aureus using the agar method. The results indicated that CNMs fabricated at laser energy 220 mJ have the highest activity against both strains and have presented inhibition zone (IZ) of about 34 ± 1.0 mm in S. aureus and 31 ± 1.5 mm for E. coli. A comparison with CNMs prepared at 160 mJ showed an IZ of 14 mm for S. aureus and of 12 mm for E. coli due to synergistic impact, while the cellular material release analysis displayed increased release with respect to the time of exposure due to inhibition of bacterial growth. [ABSTRACT FROM AUTHOR]