Biopolymers embedded with nanoparticles of metal oxides (MOs) demonstrate a wide range of bio-functions. Chitosan-incorporated MOs are an interesting class of support matrices for enhancing the biological function, compared to other support matrices. Therefore, the importance of this study lies in exploiting chitosan as a carrier not of one metal as in previous studies, but of two metals in the form of a nanocomposite to carry out several biological functions. The coprecipitation approach was employed to synthesize chitosan/Fe2O3/ZnO-nanocomposite in the present research. The characterization of chitosan/Fe2O3/ZnO-nanocomposite was performed to find out the morphology and dispersion properties of chitosan/Fe2O3/ZnO-nanocomposite. The X-ray diffraction (XRD) investigation revealed that these were crystalline. Fourier transforms infrared (FTIR) spectrum bands were viewed at 400/cm and 900/cm, due to the stretching vibration of Fe and Zn oxygen bond. TEM showed that chitosan/Fe2O3/ZnO-nanocomposite was of 20–95 nm in size. chitosan/Fe2O3/ZnO-nanocomposite exhibited inhibitory potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Candida albicans with inhibition zones of 25 ± 0.1, 28 ± 0.2, 27 ± 0.1, and 27 ± 0.2 mm, respectively while didn’t inhibited Aspergillus niger. MIC value of nanocomposite was 15.62 ± 0.33 µg/mL for C. albicans, B. subtilis and E. coli, while it was 62.50 ± 0.66 µg/mL for Pseudomonas aeruginosa. Ranged values of nanocomposite MBC (15.62 ± 0.33 to 125 ± 1 µg/mL) were attributed to all tested bacteria. Different concentrations of chitosan/Fe2O3/ZnO-nanocomposite MBC (25, 50, and 75%) reflected anti-biofilm activity against E. coli (85.0, 93.2, and 96.0%), B. subtilis (84.88, 92.21, and 96.99%), S. aureus 81.64, 90.52, and 94.64%) and P. aurogenosa (90.11, 94.43, and 98.24%), respectively. The differences in the levels of antimicrobial activities may depend on the type of examined microbes. Antioxidant activity of chitosan/Fe2O3/ZnO-nanocomposite was recorded with excellent IC50 values of 16.06 and 32.6 µg/mL using DPPH and ABTS scavenging, respectively. Wound heal by chitosan/Fe2O3/ZnO-nanocomposite was achieved with 100% compared to the untreated cells (76.75% of wound closer). The cytotoxicity outcomes showed that the IC50 of the chitosan/Fe2O3/ZnO-nanocomposite was 564.32 ± 1.46 µg/mL normal WI-38 cells. Based on the achieved findings, the chitosan/Fe2O3/ZnO-nanocomposite is a very promising agent for perform pharmacological activities.