In the present study, electrospun nanofibers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, containing natural deep eutectic solvents (NADES) were obtained and reported for the first time, exhibiting an unreported 3D morphology and enhanced charge retention properties. Choline chloride (ChCl)/urea/water in a molar ratio of 1:2:1 was used as the NADES model system. Electrospun nanofibers were produced from a 10 wt % solution of PHBV containing 26 wt % NADES with respect to the polymer and were deposited on different substrates, that is, aluminum foil and non-woven spunbond polypropylene (PP). The morphology and charge retention ability were characterized under different conditions and on different substrates. The attained fiber morphology for the NADES-containing mats showed an average fiber diameter of around 300 nm, whereas the pure PHBV polymer under the same conditions produced electrospun fibers of around 880 nm. However, the deposition of PHBV/ChCl/urea/water fibers resulted in a surprising macroscopic rugose 3D surface morphology made of aligned nanofibers when processed at 50% relative humidity (RH). The nanofiber grammages above which this 3D morphology, associated with NADES-induced charge retention, formed was found to be dependent on the substrate used and RH. This morphology was not seen at 20% RH nor when pure PHBV was produced. Charge stability studies revealed that PHBV/ChCl/urea/water nanofibers exhibited lasting charge retention, especially when sandwiched between spunbond polypropylene textiles. Finally, such multilayer structures containing a very thin double layer of PHBV/ChCl/urea/water fibers after corona treatment exhibited improved paraffin aerosol penetration, which was ascribed to the combination of thinner fibers and their charge retention capacity. The here-developed electrospun PHBV fibers containing NADES demonstrated for the first time a new potential application as electret filter media.