Subsistence hunting and commercial exploitation directly influence wildlife populations in many regions of Central and South America. Where prey populations are exploited, the foraging ecology of top-level predators can be effected negatively. This study assessed the food habits and prey selection of jaguar Panthera onca and puma Puma concolor within hunted and non-hunted segments of the Maya Biosphere Reserve (MBR), Guatemala. Food habits were determined from analysis of 76 jaguar and 145 puma scats collected within hunted and non-hunted areas of the MBR from February 2000 to August 2001. Diets of jaguar and puma were compared (1) within species between areas with and without hunting to evaluate effects of subsistence hunting, and (2) between species to evaluate resource partitioning between these sympatric carnivores. Origin of predator scats was determined from mitochondrial DNA, diets were determined from prey remnants found within scats, and frequency of prey in scats was compared to expected values based on prey density estimates to test the hypothesis that diets of jaguar and puma were selective. Densities of major prey species were estimated using line-transect sampling. White-lipped peccary Tayassu pecari, collared peccary Tayassu tajacu and brocket deer Mazama sp. were less abundant, and coatis Nasua nasua more abundant, in the hunted area than in the non-hunted area. Jaguar and puma in both hunted and non-hunted sites obtained similar dietary contributions from large prey to their respective diets despite differences in the abundance of these prey species. Diets of jaguar and puma, as measured by percentage biomass occurrence of prey species, did not differ between hunted and non-hunted areas. Jaguar diets were dominated by medium-sized prey, particularly armadillos Dasypus novemcinctus and coatis, in both hunted and non-hunted areas. Medium-sized mammals also were prominent in puma diets, but large mammals constituted approximately 50% of prey biomass in both hunted and non-hunted areas. Deer Odocoileus virginianus and Mazama sp. and large rodents Agouti paca and Dasyprocta punctata were the most important prey of puma. Dietary overlap between jaguar and puma in both hunted and non-hunted areas was low.