Human activities increasingly result in disturbance of wild animal populations. Behavioral responses vary according to the type, duration, frequency and intensity of disturbance. Exposure to disturbance, for example, arising from ecotourism, may promote tolerance of humans and drive a decrease in vigilance and avoidance behaviors, whereas disturbance resulting from extractive activities is typically more intense and is likely to be perceived as a threat. To understand how animals cope with land transformation and human presence, it is imperative to study their responses under different disturbance regimes. We performed behavioral sampling of groups of the giant otter, a charismatic top carnivore, exposed to ecotourism, extractive activities (mining and fishing) and control sites without disturbance to compare their responses. Foraging efficiency was impacted by fish abundance and thus was reduced in mined and fished areas. Giant otters in lakes with managed ecotourism did not show avoidance of research vessels and demonstrated reduced rates of defensive behavior compared to control lakes. Groups from lakes with fishing and gold mining showed increased avoidance, suggesting that these types of disturbance may involve more direct conflict. Our findings suggest that noninvasive disturbance such as ecotourism may promote giant otter tolerance to the presence of humans, whereas extractive activities may be perceived as riskier. Current protected area conservation strategies of strict ecotourism regulation and the maintenance of no-access oxbow lakes may ensure the maintenance of behavioral flexibility in giant otter populations. However, resource depletion and more intense human-giant otter interactions in disturbed areas may not be sustainable. Further studies are necessary to determine whether distinct disturbance regimes drive longer-term demographic effects.