Historic anthropogenic impacts affecting the distribution and ecology of Giant otters are well documented, however little is known regarding the factors affecting the current distribution of this species. Our objective was to determine the relative importance of temporal (seasonal river levels), and anthropogenic (presence of houses, boats and fishing nets) factors on the distribution of Giant otters around a sustainable-use protected area in the eastern Brazilian Amazon. We conducted a total of 6836.1. km of boat surveys to record locations of both direct and indirect Giant otter sightings along 198.9. km of waterways. To understand the importance of the factors at different spatial scales we conducted analysis using data summarized at two spatial scales: 50. km river “zones” and 5.1. km river sections. Our results showed that anthropogenic disturbances were the most important determinants of the presence of both direct and indirect signs, with Giant otters rarely detected within 40. km of the nearest town. Giant otters were present in the waterways throughout the annual water cycle (high, decreasing, low and increasing river levels), with direct observations positively related with the number of fishing nets present in the waterways. Our results suggest considerable spatial and temporal overlap between Giant otters and the activities of local fishermen which must be considered for the effective management of conservation conflicts in this rapidly developing region.