MurA and AroA are important antibacterial targets due to their essentiality in microorganisms and the absence of their respective pathways within mammals. Although much research has focussed on these enzymes, little is known about the evolution of AroA and MurA. Ancestral sequence reconstruction (ASR), a technique used to infer sequences of ancestral proteins and study their properties in the laboratory, was used to study the evolution of AroA and MurA enzymes from Bacillus. The ancestral AroA and MurA enzymes were functionally and structurally characterised and their properties compared to those of contemporary AroA and MurA. AroA and MurA from the last common ancestor (LCA) of Bacillus along with three intermediate Bacillus MurA ancestors were inferred using ASR. The reconstructed AroA and MurA ancestral enzymes show comparable kinetic properties to the contemporary enzymes. The thermal properties of Bacillus AroA LCA, Bacillus MurA LCA and the remaining three MurA ancestors were found to be moderately thermophilic. However, the contrasting thermal profiles observed for AroA, MurA and LeuB ancestral enzymes from Bacillus at the same period of time led to the hypothesis that reconstructed ancestral enzymes provide a snapshot of the evolving host at a given point in time. The exclusive inhibition of AroA and MurA by their inhibitors glyphosate and fosfomycin respectively remains unchanged for AroA LCA and MurA LCA. On the other hand, the high affinity for PEP and increased glyphosate sensitivity exhibited by AroA LCA indicate AroA LCA to be intermediate between class I (glyphosate sensitive) and class II (glyphosate insensitive) AroA. Characterisation of AroA LCA in vivo resulted in AroA LCA posing a fitness cost to the cells carrying this enzyme on a plasmid in a ΔaroA background. AroA LCA and MurA LCA remain highly structurally similar to the contemporary enzymes, with minor differences predominantly located on the protein surface.