As sentinel cells of innate immunity, macrophages deploy a range of antimicrobial responses to eliminate invading micro-organisms. Direct zinc toxicity against intracellular bacteria, initiated upon Toll-like Receptor (TLR) activation, has recently emerged as a novel antimicrobial strategy utilized by these cells. Here we show that macrophages are incredibly diverse in phenotype and function with respect to regulated antimicrobial zinc trafficking. Whereas primary human monocyte-derived macrophages (HMDM) differentiated with colony-stimulating factor (CSF-1) utilise zinc toxicity against intracellular bacteria, macrophages derived with granulocyte/macrophage colony-stimulating factor (GM-CSF) employ zinc starvation to limit microbial growth. Surprisingly, CSF-1-derived HMDM were more efficient than GM-CSF-derived HMDM at clearing intracellular E. coli, suggesting that the zinc toxicity response is particularly effective. To monitor the zinc toxicity antimicrobial response at a single-cell level, we developed novel zinc-stress reporter strains in E. coli, based on inducible expression of the zinc efflux transporter zntA that is selectively upregulated by cytotoxic zinc concentrations in E. coli. Using these bacterial strains, we show that, unlike non-pathogenic E. coli, a pathogenic strain of uropathogenic E. coli effectively evades the macrophage zinc toxicity response. Consequently, by comparison to non-pathogenic E. coli, this clinically relevant E. coli isolate displayed enhanced intramacrophage survival. Deletion of zntA or zntR from E. coli resulted in increased sensitivity to zinc, and these mutants were substantially compromised for intramacrophage survival, highlighting the critical role of the zinc toxicity pathway in macrophages. Finally, a comparison of CSF-1 HMDM and GM-CSF HMDM revealed specific host transporters linked with the zinc toxicity response, and we provide functional evidence for a role for one of these in macrophage-mediated bacterial clearance. Collectively, our studies reveal new insights into the deployment of TLR-inducible zinc toxicity by macrophages, as well as evasion mechanisms used by specific bacterial pathogens against this pathway.