| Summary: | This study targeted developing a novel Zinc oxide with alpha hematite nanoflowers (NFs)-loaded bee venom (Bv) (Bv-ZnO@αFe<sub>2</sub>O<sub>3</sub> NFs) as a bio-natural product from bees to combine both the advantages of combination magnetic properties and the antimicrobial and anti-biofilm properties on isolated coliform bacteria from the effluent of wastewater treatment plants. About 24 isolates of treated wastewater isolates were multidrug resistant (MDR). The phylogenetic grouping of <i>Escherichia coli</i> (<i>E. coli</i>) and <i>Klebsiella pneumonia</i> (<i>K. pneumonia</i>) showed that the largest group was Group A, followed by Group B2 and Group B1. Fourier transform infrared (FTIR), The X-ray diffraction (XRD), and scanning electron microscopy-energy dispersive X-ray analysis (SEM− EDX) validated the coating operation’s contact with Bv onto ZnO@αFe<sub>2</sub>O<sub>3</sub> NFs. According to high-resolution transmission electron microscopy (TEM) and selected area electron diffraction (SAED), pattern analyses for prepared nanoformulations exhibited a spherical shape of αFe<sub>2</sub>O<sub>3</sub> (~9–15 nm), and floral needle shapes with uniform distribution of size with aggregation of ZnOαFe<sub>2</sub>O<sub>3</sub> and Bv-ZnO@αFe<sub>2</sub>O<sub>3</sub> NFs around (~100–200 nm). The toxicity of Bv-ZnO@αFe<sub>2</sub>O<sub>3</sub> NFs was comparable up to 125 µg mL<sup>−1</sup>, when it reached 64.79% (IC<sub>50</sub>, 107.18 µg mL<sup>−1</sup>). The antibacterial activity showed different zones of inhibition against different isolates. The biofilm inhibitory activity of NPs and NFs showed a highly significant reduction (<i>p</i> < 0.001) in treated biofilms with ZnO@αFe<sub>2</sub>O<sub>3</sub> and Bv-ZnO@αFe<sub>2</sub>O<sub>3</sub>. In essence, ZnO@αFe<sub>2</sub>O<sub>3</sub> and Bv-ZnO@αFe<sub>2</sub>O<sub>3</sub> NFs are promising antimicrobials for inhibiting the growth and biofilm of MDR <i>E. coli</i> and <i>K. pneumonia</i> isolates, thereby, biocontrol of wastewater.
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