Leishmania is the causative parasite of a spectrum of diseases that cause significant global morbidity and mortality. Current antileishmanial drugs are limited and increasingly undermined by the emergence of multi-drug resistance. As an intracellular parasite, Leishmania depends on the host macrophage for its nutrient and energy resources. Interventions that attenuate parasite growth by modulating macrophage metabolism are thus emerging as attractive alternative therapeutic strategies. We performed a genome-wide small interfering RNA screen on a human monocyte cell line (THP1) infected with two Leishmania species and identified knockdown of fatty acid desaturase-1 decreased the intracellular growth of both species, indicating a role for long-chain unsaturated fatty acids in promoting Leishmania survival. We next generated a L. mexicana strain expressing TurboRFP to quantify parasitemia by flow cytometry. This assay was then used to screen the efficacy of a range of inhibitors and agonists of omega-3 and -6 fatty acid metabolism as putative antileishmanials. Targeting enzymes and transcription factors in this metabolic network dramatically perturbed the infection of bone marrow derived macrophages and THP1 cells. Significant reductions in parasitemia were observed with inhibition of phospholipase A2, fatty acid desaturase-2, 5-lipoxygenase, 15-lipoxygenase, peroxisome proliferator-activated receptor α (PPARα) and activation of PPARγ. Here, we established a robust and unbiased method to screen putative antileishmanials and measure intracellular Leishmania infection in vitro. Using this method, we have revealed a role for macrophage omega-3 and -6 fatty acid metabolism in Leishmania survival and begun to elucidate important host cell factors by which this may occur.