Protected regions, such as Malaysia's Panti Forest Reserve (PFR), play a vital role in preserving freshwater biodiversity. However, the absence of baseline data on stream macroinvertebrates and sediment nutrient contents presents a significant challenge. This study, conducted from August 21-24, 2022, as part of the Panti Expedition organized by the Johor State Forestry Department, aimed to investigate the current nutrient flows and stream macroinvertebrates in PFR, providing preliminary assessments. Utilizing kick and grab sampling techniques, we collected stream macroinvertebrates and benthic sediments along the entire upstream-to-downstream gradient of Pelepah Kiri and Gunung Muntahak. Our findings revealed a diverse assemblage of 385 macroinvertebrates from 28 distinct families, with notable abundance observed in Baetidae (14.0%), Simuliidae (10.6%), and Hydropsychidae (10.6%). The analysis of overall taxonomic composition indicated a Jaccard index similarity of 0.64, suggesting overlapping communities between the two sites. Predators were significantly more abundant in Pelepah Kiri, while collectors were more abundant in Gunung Muntahak. The expedition trails exhibited varying levels of moisture content (ranging from 17.2% to 31.3%), total organic carbon content (≤ 0.4%), and total nitrogen levels (ranging from 1,330 mg/kg to 3,180 mg/kg), while phosphorus loading remained undetectable due to low concentration levels. Nutrient analyses indicated no significant impact of total nitrogen on macroinvertebrate family richness, abundance, and diversity. The study's biological metrics demonstrated good stream quality, with taxa richness ranging from 21 to 20 and EPT taxa richness ranging from 6 to 10. These results highlight the diverse freshwater macroinvertebrate communities in PFR, suggesting the maintenance of favorable water quality for biodiversity preservation. Overall, this study provides critical baseline data for future conservation management strategies in PFR, contributing to our understanding of the intricate connections between nutrient dynamics and stream macroinvertebrates in protected areas.