Development of eco-friendly and biodegradable package materials is an important goal of modern science and international industry. Poly(lactic)-co-glycolic acid (PLGA) is suitable for this purpose. However, biocompatible materials may be contaminated with bacteria. This problem may be solved by the addition of metal oxides nanoparticles (NPs) with antibacterial properties. Although metal oxides NPs often show cytotoxicity against plant and mammalian cells, a new nanocomposite based on PLGA and aluminum oxide (Al2O3) NPs has been developed. The PLGA/Al2O3 NP composite has pronounced antibacterial properties. The addition of Al2O3 NPs 0.01% inhibited growth of E. coli for >50%. The antimicrobial effect of Al2O3 NPs is implemented through the generation of reactive oxygen species and damage of bacterial proteins and DNA. The biocompatibility of the nanocomposite with plant and mammalian cells was studied. The PLGA/Al2O3 NP composite did not influence the growth and development of tomatoes and cucumbers. PLGA and its composite with Al2O3 NPs 0.001–0.1% did not influence viability and proliferation of mammalian cells, on their density or substrate colonization rate. The developed nanocomposite has controlled mechanical properties, high antibacterial activity and high biocompatibility, which makes it an attractive candidate for building and food package material manufacture and agriculture.