The effects of benzalkonium chloride (BKC) contents on the structure, properties, and ultrafiltration performance of chitosan-based nanocomposite membranes containing poly(ethylene glycol) and multi-walled carbon nanotube (chitosan/BKC/PEG/CNT) were examined. The membranes were prepared by a mixing solution method and phase inversion before being characterized with microscopic techniques, tensile tests, thermogravimetric analysis, water contact angle, and porosity measurements. The performance of the nanocomposite membranes in regard to permeability (flux) and permselectivity (rejection) was examined. The results show that the incorporation of BKC produced nanocomposite membranes with smaller pore structures and improved physico-chemical properties, such as an increase in porosity and surface roughness (Ra = 45.15 to 145.35 nm and Rq = 53.69 to 167.44 nm), an enhancement in the elongation at break from 45 to 109%, and an enhancement in the mechanical strength from 31.2 to 45.8 MPa. In contrast, a decrease in the membrane hydrophilicity (water contact angle increased from 56.3 to 82.8°) and a decrease in the average substructure pore size from 32.64 to 10.08 nm were observed. The membrane rejection performances toward Bovine Serum Albumin (BSA) increased with the BKC composition in both dead-end and cross-flow filtration processes. The chitosan/BKC/PEG/CNT nanocomposite membranes have great potential in wastewater treatments for minimizing biofouling without reducing the water purification performance.