Atovaquone is an approved antimalarial drug which could sensitise tumours to radiation and may modulate the tumour immune microenvironment. Here we evaluated the triple combination of radiation with atovaquone plus anti-PD-L1 in HNSCC syngeneic mouse tumour models. We observed significant tumour growth delay induced by the triple combination treatment with differential responses in three different mouse models. There was complete tumour eradication in 6 of 12 mice in the MOCL1 model but not in the other two murine models. The triple combination was associated with an increased population of CD8+-T Granzyme B+ cells as well as decreased populations of Tregs, CD11b+Gr1+ granulocytic MDSCs and CD11b+ Ly.6G+ neutrophils. Atovaquone inhibited EGFR and its downstream signalling as well as reduced PD-L1 expression. The differential antitumour effects of the triple combination may be due to different basal and post-treatment levels of PD-L1, pERK1/2 and pS6, in particular, their levels were reduced by atovaquone ± RT in MOCL1 models and not in the other two murine models. Radiotherapy plus atovaquone combination increased cell cycle arrest in G2/M phase, which led to inhibition of DNA damage repair by homologous recombination and increased cell death. We also observed increased activation of CD8+-T cells that led to enhanced killing in our HNSCC patient-derived organoid models treated with the triple combination of radiotherapy and atovaquone plus PD-1/PD-L1 blockade. Overall, our results suggest that the triple combination of radiotherapy and atovaquone plus PD-1/PD-L1 blockade has the potential to increase the efficacy of radiotherapy and/or immunotherapy. Thus, this may provide a rationale to further validate this triple combination of radiotherapy and atovaquone plus PD-1/PD-L1 blockade in clinical trials to improve treatment outcomes.