Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP). Drug treatment failure has been associated epidemiologically with point mutations in the gene for dihydropteroate synthase which is part of a gene that encodes three covalently linked enzymes involved in folic acid synthesis (FAS). The evaluation of whether mutations found in P. jirovecii FAS lead to sulfa drug resistance is hampered by the lack of a culture system for P. jirovecii as well as the failure of P. jirovecii FAS to complement in a heterologous system. Therefore, we chose to model the P. jirovecii mutations in the Saccharomyces cerevisiae FAS protein (encoded by FOL1) via its expression in Escherichia coli. An optimized drug diffusion assay was used to evaluate the FAS mutants against 15 sulfa drugs. It was established that the single amino acid substitution, P₅₉₉S, in the (DHPS) domain of FAS led to sulfa drug resistance, whereas the T₅₉₇A substitution led to increased sensitivity. The presence of both mutations (T₅₉₇A and P₅₉₉S) was cooperative and led to increased sulfa drug resistance. Analysis of a novel double mutant, (T₅₉₇V P₅₉₉S) was found to have significantly higher sulfa drug resistance than the T₅₉₇A P₅₉₉S mutant. These data suggest that further amino acid substitutions may lead to the evolution of higher sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine and sulfathiazole) were identified that had higher inhibitory potential than sulfamethoxazole, which is currently the preferred treatment for PCP.