This study focuses on evaluating the efficacy of biofilm-enhanced natural zeolite for the purification of slaughterhouse wastewater. The investigation encompasses four distinct treatment methods: employing natural zeolite without biofilm, integrating biofilm into 1–2 mm particle size natural zeolite, enhancing biofilm in less than 4 mm particle size natural zeolite, and introducing biofilm in less than 8 mm particle size natural zeolite. The outcomes underscore the substantial improvement brought about by biofilm incorporation. For instance, within the natural zeolite treatment system without biofilm, the final effluent retained 28 NTU of turbidity. In contrast, utilizing the <8 mm particle size with biofilm resulted in 3.2 NTU of turbidity in the treated effluent, 2.45 NTU for the <4 mm particle size with biofilm, and 1.02 NTU for the 1–2 mm particle size zeolite system with biofilm. Notably, the achieved removal rates were significant, reaching 79.88% for natural zeolite without biofilm, 97.69% for the <8 mm particle size with biofilm, 99.27% for the <4 mm particle size with biofilm, and 98.24% for the 1–2 mm particle size zeolite system with biofilm. It is noteworthy that the removal efficiencies varied from 50 to 100% for wastewater samples subjected to the treatment system without biofilm, 65.7–100% with the <8 mm particle size biofilm, 71.4–100% with the <4 mm particle size biofilm, and 71.7–100% with the 1–2 mm particle size zeolite system biofilm. These findings collectively emphasize the pivotal role of biofilm in enhancing treatment outcomes, presenting a promising avenue for optimizing wastewater treatment efficiency.