The Bonny Estuary, located in the Niger Delta, represents one of the region’s most ecologically and economically significant aquatic systems, comprising a network of creeks and tributaries that support diverse habitats. These ecosystems, however, are increasingly exposed to pollution, which can significantly alter species abundance and composition. Benthic macrofauna—organisms living on or within the sediment—serve as critical ecological indicators due to their habitat specificity, limited mobility, and direct exposure to environmental stressors. Their close association with sediments makes them particularly sensitive to pollutants, providing early-warning signals of ecosystem degradation. This study investigates variations in benthic macrofaunal assemblages in the Upper Bonny Estuary in relation to different sieve mesh sizes used during sampling. By evaluating how mesh size influences the recovery of macrofaunal species, the research aims to determine the accuracy and reliability of benthic assessments. The findings highlight that sieve mesh selection can significantly affect species richness, abundance, and diversity, underscoring the importance of standardized sampling protocols for ecological monitoring. Moreover, the study reinforces the value of benthic macroinvertebrates as bio-indicators for detecting pollution and assessing the overall health of estuarine environments. The results contribute to improved methodology in benthic ecological studies and offer insights for sustainable management and conservation of the Bonny Estuary. They demonstrate that careful consideration of sampling techniques, alongside regular monitoring of benthic communities, is essential for detecting early ecological changes and mitigating the impacts of anthropogenic and environmental stressors.