Healthcare-associated infections remain a significant concern, driving the exploration of metal-based nanosystems as innovative solutions for developing antimicrobial polymer membranes. This study evaluates the toxicity of a novel nanofiber membrane reinforced with multimetallic silica nanohybrids, proposed as an advanced antimicrobial layer. Initially, silver (Ag), copper (Cu), and cobalt (Co) were doped into silica nanoparticles (SiNPs) the sol-gel method. The trimetallic nanohybrid demonstrated superior antimicrobial activity compared to Ag-, Cu-, or Co-doped SiNPs alone, showing enhanced efficacy against Gram-positive and Gram-negative bacteria, as well as fungi, due to the synergistic action of the metals. Toxicity was assessed using zebrafish embryo assays (OECD 236) with concentrations ranging from 10.000 to 0.156 mg L. LC values were 2.05 mg L (AgSiNPs), 5.53 mg L (CuSiNPs), 9.99 mg L (CoSiNPs), and 6.35 mg L (trimetallic SiNPs). Based on these results, the trimetallic nanohybrid, which showed the lowest toxicity, was electrospun into a polymer membrane. Skin irritation was evaluated using the HET-CAM assay (ICCVAM protocol), yielding irritation scores of 13.33 ± 0.58 (Ag), 11.67 ± 0.58 (Cu), 1.00 ± 1.00 (Co), and 3.67 ± 0.58 (trimetallic), suggesting minimal irritation potential ( = 0.05). Franz diffusion cell analysis detected no Cu or Co, and only 0.26 mg L of Ag after 24 hours, well below toxic thresholds. Statistical analysis ( < 0.05) confirmed the safety of the membrane, showing reduced toxicity and minimal metal nanohybrid diffusion. The multimetallic nanohybrid membrane is therefore considered safe as an antimicrobial polymer membrane in healthcare applications and recommends further evaluation.