Owing to their unique properties, zinc oxide nanoparticles (ZnO NPs) have a broad range of exciting applications. However, the problem of nanoparticles aggregation remains challenging. So, biopolymers of polysaccharides can provide green and promising stabilizers as alternatives to the current toxic chemical stabilizers during ZnO NPs synthesis. The main idea in this investigation is to tune ZnO NPs with an appropriate texture, shape, and size for antibacterial application. So, this work compares the use of three different eco-friendly stabilizers namely starch, carboxymethyl cellulose, and hydroxyethyl cellulose as alternatives capping agents in the fabrication of ZnO NPs at various times. The optimized ZnO NPs sample was obtained using starch as the optimum stabilizer at reaction conditions of 0.5 h, room temperature (25 °C), 1:2) MZn:MNaOH) ratio and 1% (w/v) starch concentration. The optical, texture, and structural properties of prepared ZnO NPs were characterized by UV–Vis, DLS, zeta potential, FT-IR, and TEM techniques. ZnO NPs showed a mean zeta potential of −21.6 mV, explaining that they are moderately stable. The analysis by TEM confirmed that the NPs were spherical and have an average size of 23 nm. The antibacterial properties of ZnO NPs against Gram-positive (Bacillus subtilis and Staphylococcus epidermidis) and Gram-negative (Enterobacter cloacae and Escherichia coli) bacteria were evaluated based on the zone of inhibition (ZOI) values expressed in mm. The results showed promising performances for their antibacterial activity against the tested bacteria which indicated a strong antibacterial activity of ZnO NPs against B. subtilis, S. epidermidis, and E. cloacae with ZOI values of 17, 14 and 16 mm, respectively, and it showed moderate activity against E. coli (ZOI = 10 mm). The synthesis of biopolymer stabilized ZnO NPs by this approach could be eco-friendly and cost-effective and synthesized ZnO NPs can serve as promising antibacterial agents.