Freshwater resources underpin multiple livelihood systems around the world, particularly in highly productive tropical floodplain regions. Sustaining Indigenous people’s access to freshwater resources for customary harvesting, while developing alternative livelihood strategies can be challenging. The sustainable livelihoods approach was applied to examine the ways in which multiple livelihoods in the East Alligator River floodplain region in northern Australia influence Aboriginal people’s access to freshwater resources for customary harvesting. Interviews with Aboriginal residents were conducted to understand changes to freshwater customary harvesting practices. The dominant floodplain-based livelihoods analysed were pastoralism, biodiversity conservation and tourism and they were found to generate both opportunities and constraints for sustaining freshwater customary harvesting. Opportunities were provided through facilitating regular access to floodplain country and opportunistic access for harvesting, which assists in sustaining bio-cultural knowledge. Partnerships developed through these mainstream livelihoods built human capacity that enhanced all livelihood resource capitals (natural, human, social, financial and physical). Three key ways the dominant livelihoods constrained access to key freshwater resources were identified. Tourism required sacrificing certain hunting places and had to accommodate recreational fishing pressure. The successful recovery of the saltwater crocodile population through biodiversity conservation policy has inadvertently reduced people’s customary access to in-stream resources. Pastoralism on the floodplain had restricted traditional floodplain burning practices associated with accessing aestivating long-necked turtles, affecting access and abundance. These findings highlight the need for the development of remote Indigenous livelihood strategies to make explicit their influences on freshwater customary harvesting practices, to better support their maintenance amongst multiple, non-customary floodplain livelihoods.