Background/Objectives: Bee venom (BV), as a natural product, is one of the foundations of the pharmaceutical industry, through which many diseases, including serious ones, can be effectively treated. The BV nanofilm is an effective antidote delivered into the human body to target the affected area and address the issue without major side effects. In this study, we investigated the intriguing therapeutic effects of apitoxin (bee venom) used in isolation, combined with the powerful properties of zinc oxide nanoparticles. Methods and Results: BV nanofilm was evaluated using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The BV extract was analyzed using mass spectrometry (MS), which identified 84 active components present at varying concentrations. BV was treated with both polyvinyl alcohol (PVA) and zinc oxide nanoparticles (ZNPs) to increase the intensity of OH and CH2 groups and to enhance the dispersion of C=O. BV has demonstrated anti-type 2 diabetes activity by inhibiting α-amylase and α-glucosidase, which are starch-degrading enzymes. The nanofilm is an active mixture of BV, PVA, and ZNPs, which exhibited the highest antidiabetic activity with IC50 values of 30.33 μg/mL and 5.55 μg/mL for the inhibition of α-amylase and α-glucosidase, compared to IC50 of 51.69 µg/mL and IC50 of 7.30 µg/mL for BV, respectively. The nanofilm also showed higher anti-inflammatory activity by inhibiting red blood cell (RBC) hemolysis, with an IC50 of 16.99 μg/mL in comparison to IC50 of 72.99 µg/mL for BV alone. The nanofilm demonstrated broad-spectrum antimicrobial activity, effectively targeting both Gram-positive (Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633) and Gram-negative bacteria (Salmonella typhi ATCC 6539, Escherichia coli ATCC 8739). Furthermore, increased antioxidant activity was recorded by inhibiting the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging effect with an IC50 of 4.26 μg/mL and 19.43 μg/mL for nanofilm and BV, respectively. BV was found to be more toxic to liver tissue (HepG2 cell line) than nanofilm, with IC50 values of 18.5 ± 0.08 μg/mL and 52.27 ± 0.7 μg/mL, respectively. The BV extract displayed higher toxicity to liver tissue (2.3%) with 97.7% viability at 250 μg/mL, compared to nanofilm, which showed 0.09% toxicity and 99.9% viability at the same concentration. Conclusions: the BV nanofilm emerges as a promising alternative medicine, offering an innovative solution for treating various diseases through its high concentration of therapeutically active compounds and effortless targeting delivery.