Consolidated bioprocessing (CBP) of lignocellulosic biomass offers an alternative route to renewable energy. The crop pathogen Fusarium oxysporum is a promising fungal biocatalyst because of its broad host range and innate ability to co-saccharify and ferment lignocellulose to bioethanol. A major challenge for cellulolytic CBP-enabling microbes is alcohol inhibition. This research tested the hypothesis that Agrobacterium tumefaciens - mediated transformation (ATMT) could be exploited as a tool to generate phenotypic diversity in F. oxysporum to investigate alcohol stress tolerance encountered during CBP. A random mutagenesis library of gene disruption transformants (n=1,563) was constructed and screened for alcohol tolerance in order to isolate alcohol sensitive or tolerant phenotypes. Following three rounds of screening, exposure of select transformants to 6% ethanol and 0.75% n-butanol resulted respectively in increased (≥11.74%) and decreased (≤43.01%) growth compared to the wild -type (WT). Principal component analysis (PCA) quantified the level of phenotypic diversity across the population of genetically transformed individuals and isolated candidate strains for analysis. Characterisation of one strain, Tr. 259, ascertained a reduced growth phenotype under alcohol stress relative to WT and indicated the disruption of a coding region homologous to a putative sugar transporter (FOXG_09625). Quantitative PCR (RT-PCR) showed FOXG_09625 was differentially expressed in Tr. 259 compared to WT during alcohol-induced stress (P<0.05). Phylogenetic analysis of putative sugar transporters suggests diverse functional roles in F. oxysporum and other filamentous fungi compared to yeast for which sugar transporters form part of a relatively conserved family. This study has confirmed the potential of ATMT coupled with a phenotypic screening program to select for genetic variation induced in response to alcohol stress. This research represents a first step in the investigation of alcohol tolerance in F. oxysporum and has resulted in the identification of several novel strains, which will be of benefit to future biofuel research.