Isoporous membranes with high pore density and sharp pore size distribution in the micro- and nanoscale providing effective separation are necessary to enable advances in different biological and medical technologies for applications such as detection of cancer cells or pathogens, isolation of circulating tumor cells for blood purification or diagnostic analysis purposes, DNA or proteins sensing, controlled drug delivery or microfluidic devices for organ-on-chip, as well as investigations in plant and bioscience. Hereby, we propose two nanotechnology methods, both combined with lithography and dry reactive ion etching processes, to fabricate a series of polyester membranes with isopores of size in microscale (0.7 to 50 µm) and in nanoscale (300 nm), which demonstrated high porosity and surface area. The membranes fabrication methodologies are environmentally friendly from the aspect that they do not require solvents, and, without water waste characteristics. The membranes are flexible, have excellent chemical and thermal resistance as well as biocompatibility characteristic. All membranes have pore density 10-fold higher than track-etched analogues and exhibit much higher permeance for both microporous and nanoporous membranes. The isoporous system in micron and submicron scales was successfully tested for different applications. The first one was the mild fractionation of organelles of Arabidopsis homogenates. The success indicates that the membranes could be potentially extended to other biological fractionations in substitution of more tedious centrifugation steps. The second successfully demonstrated application is air purification. The membranes had a particulate matter capture efficiency as high as 99 %. In this case, they were coated with silver nanoparticles, which provided high antibacterial effect. The nano-isoporous membranes were successfully demonstrated for nanoparticles sieving. Moreover, by extending the methods to the fabrication of polyimide isoporous membranes, we have proved that they can be easily adapted to other homopolymeric materials that can be chosen according to the needed balance of required stability and economic factors. As far as the resolution is concerned, the achievements we report are among the best combinations of isoporosity with small pore sizes, high pore density and large total active filtration area, currently feasible to fabricate.