The clinical use of photodynamic therapy (PDT) with rose bengal (RB) is emerging as an effective t reatment for a range of applications given its non-invasive and localised mode of delivery. In particular, rose bengal PDT has shown promising antifungal action <i>in vitro</i>. While focus has largely been on the physical and chemical impacts of PDT on the cell, an understanding of the role of genetics underpinning the cellular response is still limited. We have, therefore, reported a screen of the entire non-essential gene library of the model organism, <i>Saccharomyces cerevisiae</i>, using rose bengal PDT to ascertain the key genetic pathways affecting fungal tolerance to PDT. We also investigated the dosage of PDT required to eradicate <i>Trichophyton rubrum</i> spores, the main causative organism of onychomycosis infection. Following this, we conducted a pilot patient study of six patients (seven toenails) for the treatment of onychomycosis using rose bengal PDT (140 μM RB and ~763 J/cm<sup>2</sup> green light), where the clinical treatment protocol was developed on the basis of the <i>in vitro</i> outcomes. The key biochemical pathways identified by the genetic screen as having altered tolerance to PDT included ergosterol biosynthesis, vacuolar acidification, and purine/S-adenosyl-L-methionine biosynthesis. The subsequent pilot patient study saw the complete cure of onychomycosis for all patients within three to five treatment sessions in the absence of pain or other local side effects. The outcome of the genetic screen for tolerance may thus inform the development of efficient clinical treatments using rose bengal PDT.