This study targeted developing a novel Zinc oxide with alpha hematite Nano flowers (NFs)-loaded bee venom (Bv) (Bv-ZnO@αFe2O3 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 Escherichia coli (E. coli) and Klebsiella pneumonia (K. pneumonia) 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@αFe2O3 NFs. According to high-resolution transmission electron microscopy (TEM) and selected area electron diffraction (SAED), pattern analyses for prepared Nano formulations exhibited a spherical shape of αFe2O3 (~9–15 nm), and floral needle shapes with uniform distribution of size with aggregation of ZnOαFe2O3 and Bv-ZnO@αFe2O3 NFs around (~100–200 nm). The toxicity of Bv-ZnO@αFe2O3 NFs was comparable up to 125 µg mL−1, when it reached 64.79% (IC50, 107.18 µg mL−1). The antibacterial activity showed different zones of inhibition against different isolates. The biofilm inhibitory activity of NPs and NFs showed a highly significant reduction (p < 0.001) in treated biofilms with ZnO@αFe2O3 and Bv-ZnO@αFe2O3. In essence, ZnO@αFe2O3 and Bv-ZnO@αFe2O3 NFs are promising antimicrobials for inhibiting the growth and biofilm of MDR E. coli and K. pneumonia isolates, thereby, biocontrol of wastewater.