The overuse of antimicrobials in medicine and agriculture has led to the emergence of antimicrobial resistant bacteria, which are difficult to treat and incur significant healthcare costs. The purpose of this thesis was to characterize important antimicrobial resistance genes (fosA and mmr) from two organisms identified as pathogens of concern by the World Health Organization; Escherichia coli and Mycobacterium tuberculosis. The goal of these two studies was to distinguish the antimicrobial resistant phenotypes of these genes to clinically relevant antimicrobial agents and learn more about their drug selectivity, their structure-function, and their phylogenetic relationships to previously characterized representative homologs. The first objective was to characterize the fosfomycin resistance (fosA) genes from three Canadian clinical E. coli isolates with high-level fosfomycin resistance (MIC >512 µg/mL). Whole genome sequencing was performed on these isolates which uncovered fosA3, fosA8, and a novel gene fosA7.5. The fosA3, fosA8, and three fosA7.5 variants (fosA7.5WT, fosA7.5Q86E, fosA7.5W92G) were individually cloned and over-expressed in E. coli K-12 BW25113. Antimicrobial susceptibility testing using Clinical and Laboratory Standards Institute (CLSI) methodology was performed to confirm the role of these fosA genes in conferring fosfomycin resistance. Each gene was found to confer high level fosfomycin resistance, with the exception of fosA7.5W92G which did not confer resistance to fosfomycin. Phylogenetic comparison, protein sequence alignment, and homology modelling of the FosA7.5 variants identified amino acid residues that distinguish this sub-family and play an important role in the active site of these enzymes. The second objective was to characterize the small multidrug resistance (SMR) family efflux gene mmr (Rv3065) from M. tuberculosis H37Rv using M. smegmatis as a model organism for expression. The mmr gene was electroporated into M. smegmatis mc24517, and compared to eight other representative SMR genes (emrE, qacE, qacF, gdx, qacC, qacG, qacH, and qacJ) which were cloned in either E. coli K-12 BW25113 or B. subtilis MGNA-A001. CLSI broth microdilution antimicrobial susceptibility was used to determine substrate specificity differences between over-expressed efflux pumps. Only mmr was shown to confer 2-4 fold reduced susceptibility to erythromycin, clarithromycin, azithromycin, roxithromycin, and clindamycin. Phylogenetic analysis, amino acid sequence alignments, and homology modelling of Mmr to previously characterized SMR family representatives identified several conserved regions and multiple residue differences that may explain the differences in Mmr macrolide selectivity.