The clinical success of linezolid for treating Gram-positive infections paired with the high conservation of bacterial ribosomes predicts that if oxazolidinones were engineered to accumulate in Gram-negative bacteria, then this pharmacological class would find broad utility in eradicating infections. Here, we report an investigative study of a strategically designed library of oxazolidinones to determine the effects of molecular structure on accumulation and biological activity. <i>Escherichia coli</i>, <i>Acinetobacter baumannii</i>, and <i>Pseudomonas aeruginosa</i> strains with varying degrees of compromise (in efflux and outer membrane) were used to identify motifs that hinder permeation across the outer membrane and/or enhance efflux susceptibility broadly and specifically between species. The results illustrate that small changes in molecular structure are enough to overcome the efflux and/or permeation issues of this scaffold. Three oxazolidinone analogues (<b>3e</b>, <b>8d</b>, and <b>8o</b>) were identified that exhibit activity against all three pathogens assessed, a biological profile not observed for linezolid.