Janus nanofiber based composite ultrafiltration (UF) membranes were fabricated via a two-step method, i.e., consecutive electrospinning of hydrophilic nylon-6,6/chitosan nanofiber blend and conventional casting of hydrophobic poly(vinylidene difluoride) (PVDF) dope solution. The as-developed PVDF/nylon-6,6/chitosan membranes were investigated for its morphology using Scanning Electron Microscopy (SEM) by which 18 wt % PVDF was chosen as the optimum base polymer concentration due to optimal degree of integration of cast and nanofiber layers. This membrane was benchmarked against the pure PVDF and PVDF/nylon-6,6 membranes in terms of surface properties, permeability, and its ability to reverse protein fouling. The improved hydrophilicity of the PVDF/nylon-6,6/chitosan membrane was revealed from the 72% reduction in the initial water contact angle compared to the pure PVDF benchmark, due to the incorporation of intrinsic hydrophilic hydroxyl and amine functional groups on the membrane surface confirmed by FTIR. The integration of the nanofiber and cast layers has led to altered pore arrangements offering about 93% rejection of bovine serum albumin (BSA) proteins with a permeance of 393 L·m·h·bar in cross-flow filtration experiments; while the PVDF benchmark only had a BSA rejection of 67% and a permeance of 288 L·m·h·bar. The PVDF/nylon-6,6/chitosan membrane exhibited high fouling propensity with 2.2 times higher reversible fouling and 78% decrease in the irreversible fouling compared to the PVDF benchmark after 4 h of filtration with BSA foulants.