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Abstract While numerous environmental factors contribute to the spread of antibiotic resistance genes (ARGs), quantifying their relative contributions remains a fundamental challenge. Similarly, it is important to differentiate acute human health risks from environmental exposure, versus broader ecological risk of ARG evolution and spread across microbial taxa. Recent studies have proposed various methods for achieving such aims. Here, we introduce MetaCompare 2.0, which improves upon original MetaCompare pipeline by differentiating indicators of human health resistome risk (potential for human pathogens of acute resistance concern to acquire ARGs) from ecological resistome risk (overall mobility of ARGs and potential for pathogen acquisition). The updated pipeline's sensitivity was demonstrated by analyzing diverse publicly-available metagenomes from wastewater, surface water, soil, sediment, human gut, and synthetic microbial communities. MetaCompare 2.0 provided distinct rankings of the metagenomes according to both human health resistome risk and ecological resistome risk, with both scores trending higher when influenced by anthropogenic impact or other stress. We evaluated the robustness of the pipeline to sequence assembly methods, sequencing depth, contig count, and metagenomic library coverage bias. The risk scores were remarkably consistent despite variations in these technological aspects. We packaged the improved pipeline into a publicly-available web service (http://metacompare.cs.vt.edu/) that provides an easy-to-use interface for computing resistome risk scores and visualizing results.