Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance. Members of the major facilitator superfamily (MFS) are more prevalent than the ATP-binding cassette (ABC) multidrug transporters among Saccharomyces cerevisiae and clinically important fungi. Even though fungi can withstand various environmental selective pressures due to their ecological, morphological, and biological plasticity, there is no established association of antifungal resistance with fungal mitochondrial MFS. Here, we have hypothesized that fungal mitochondria will contain efflux pumps of the major facilitator superfamily (MFS) that could participate in antimicrobial resistance. We are interested in investigating this phenomenon in fungal mitochondria. In one approach, we have cloned and expressed a set of S. cerevisiae transporters of the MFS namely Transporter of Polyamines (Tpo1 to Tpo5) and Hexose transporter (Hxt5), some of which are localized in mitochondria, in an Escherichia coli expression system. Following optimization of expression in E. coli, we have carried out the functional analysis of these putative pumps to determine the specific substrates using mitochondria-targeting antibiotics. We have shown chloramphenicol (CAP) and erythromycin (ERY) as potential substrates of Tpo1 and Tpo3 using indirect assays (antibiotic susceptibility testing). However, we were unable to establish if these proteins were efflux pumps using a direct ethidium bromide (EtBr) accumulation assay. In another approach, we have examined S. cerevisiae BY4741 strains that each are missing one of the pumps from Tpo1 to Tpo4. We tested if the absence of a pump causes increased susceptibility to antibiotics. We have shown that Tpo3 is likely involved in protecting yeast cells from CAP and ERY, and this is supported by qualitative results measuring growth phenotype and quantitative results of growth kinetics. Finally, we could demonstrate evidence for efflux of EtBr by Neurospora crassa wild-type (WT) mitochondria, but EtBr efflux could not be shown for S. cerevisiae BY4741 mitochondria. We anticipate our study is a starting point for future in-vivo models to look into mitochondrial drug accumulation and efflux-mediated resistance in fungi.