Following the advent of industrial-scale antibiotic production in the 1940s,1 antimicrobial resistance (AMR) has been on the rise and now poses a major global health threat in terms of mortality, morbidity, and economic burden.2,3 Because AMR can be exchanged between humans, livestock, and wildlife, wild animals can be used as indicators of human-associated AMR contamination of the environment.4 However, AMR is a normal function of natural environments and is present in host-associated microbiomes, which makes it challenging to distinguish between anthropogenic and natural sources.4,5 One way to overcome this difficulty is to use historical samples that span the period from before the mass production of antibiotics to today. We used shotgun metagenomic sequencing of dental calculus, the calcified form of the oral microbial biofilm, to determine the abundance and repertoire of AMR genes in the oral microbiome of Swedish brown bears collected over the last 180 years. Our temporal metagenomics approach allowed us to establish a baseline of natural AMR in the pre-antibiotics era and to quantify a significant increase in total AMR load and diversity of AMR genes that is consistent with patterns of national human antibiotic use. We also demonstrated a significant decrease in total AMR load in bears in the last two decades, which coincides with Swedish strategies to mitigate AMR. Our study suggests that public health policies can be effective in limiting human-associated AMR contamination of the environment and wildlife.