Candida albicans uniquely possesses an expanded family of genes (the TLO gene family) that encodes 10-15 paralogues of the Med2 component of the transcriptional regulator Mediator. Previous studies have shown that TLO null mutants are unable to form hyphae and are hypersensitive to environmental stress. However, the reason for the TLO gene expansion remains unclear, and the current study aimed to determine if reduction in the TLO family copy number affected virulence. In order to investigate this, we used CRISPR-Cas9 mutagenesis to generate two TLO-depleted mutants: one mutant retaining only TLOβ2 (CaTLO2) and the second mutant containing only TLOγ5 (CaTLO5). Both TLO-depleted mutants exhibited increased filamentous growth, increased susceptibility to specific stresses and reduced virulence in a murine model of oropharyngeal candidiasis (OPC). In vitro, the CaTLO5 mutant also exhibited impaired hyphal escape from macrophages and reduced hyphal invasion of oral keratinocytes. We then investigated if complementation with TLOα1, a gene previously shown to restore wild-type growth in a Δtlo null mutant, could restore virulence. In vitro infection models showed that TLOα1 could restore true hypha formation, epithelial invasion and hyphal escape from macrophages in the CaTLO5 background. The murine OPC model showed that TLOα1 could restore wild-type virulence in both CaTLO2 and CaTLO5 strains, suggesting an essential role for α-TLO in oral mucosal infection. Together, these findings highlight the functional specialization between the α, β and γ TLO gene groups and establish α-TLO as a major regulator of virulence in C. albicans.