These days, composite materials based on polymers and inorganic nanoparticles (NPs) are widely used in optoelectronics and biomedicine. In this work, composite membranes of polylactic acid and ZnO NPs containing 5–40 wt.% of the latter NPs were produced by means of electrospinning. For the first time, polymer material loaded with up to 40 wt.% of ZnO NPs (produced via laser ablation in air and having non-modified surface) was used to prepare fiber-based composite membranes. The morphology, phase composition, mechanical, spectral and antibacterial properties of the membranes were tested by a set of analytical techniques including SEM, XRD, FTIR, UV-vis, and photoluminescence spectroscopy. Antibacterial activity of the materials was evaluated following standard procedures (ISO 20743:2013) and using S. aureus and E. coli bacteria. It is shown that incorporation of 5–10 wt.% of NPs led to improved mechanical properties of the composite membranes, while further increase of ZnO content up to 20 wt.% and above resulted in their noticeable deterioration. At the same time, the antibacterial properties of ZnO-rich membranes were more pronounced, which is explained by a larger number of surface-exposed ZnO NPs, in addition to those embedded into the bulk of fiber material.