Pressure injuries (PIs) are interruptions in the skin that cause great discomfort in bedridden patients and their treatment generates a lot of waste and equal discomfort, requiring alternative materials for their treatment. Thus, this work aims to synthesize and characterize copper nanoparticles (CuNPs) by the biosynthesis method from ascorbic acid for possible application in tissue engineering. CuNPs were characterized by X-ray diffraction (XRD), N2 adsorption/desorption, zeta potential (ZP), Attenuated Total Reflection Fourier Transform Infrared (ATR-FT-IR) spectroscopy, Field Emission Gun Scanning Electron Microscopy (FEG-SEM) and Scanning Electron Microscopy with Energy Dispersive X-Ray Analysis (SEM-EDX). The safety profile was performed in vitro cytotoxicity MTT assay in the Vero cell line at different time intervals (24, 48 and 72 h). The antibacterial potential was evaluated by minimal inhibitory activity (MIC) against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 13883) and Klebsiella pneumoniae (ATCC 27853). The XRD diffractogram showed characteristic peaks of CuNPs with a cubic face structure representing metallic copper at 43.31° (111) plane with an average particle size of 43.6 nm. The micrograph of CuNPs showed heterogeneous clusters, with SBET = 8 ± 0.05 m2 g−1, Vp = 0.04 ± 0.02 cm³ g−1 and ZP = −29.63 ± 2.31 mV and H2-type hysteresis. EDX results indicated the presence of CuNPs and the effectiveness of the biosynthesis process. The safety profile showed that 12.5 μg mL−1 was the maximum concentration accepted by cells, and that the damage caused in Vero cells was due to the generation of reactive oxygen species after 72 h. Regarding antimicrobial activity, CuNPs show greater inhibition and selectivity for gram-negative bacteria, such as P. aeruginosa (0.625 mg mL−1), K. pneumoniae (0.156 mg mL−1) and E. coli (0.625 mg mL−1) due to the greater affinity of Cu+2 ions with the structure of the cell wall. Therefore, to highlight the potential of CuNPs synthesized from ascorbic acid by biosynthesis for a possible application in tissue engineering.