Mitochondrial missense mutations and pathogenic variants have been implicated in the pathogenesis of COVID‐19. This study evaluated the role of mitochondrial DNA (mtDNA) mutations and changes in gene expression in the progression of COVID-19 and their correlation with clinical characteristics. Next‐generation sequencing with high throughput was used to identify mtDNA mutations in 30 COVID-19 patients compared to 20 healthy controls. The potential impact of identified mutations on protein structure and stability was predicted using bioinformatic tools. Quantitative real-time polymerase chain reaction was employed to assess the expression levels of mtDNA-encoded genes involved in oxidative phosphorylation in COVID-19 patients and healthy controls. Correlations between gene expression levels, clinical parameters, including leukocyte, lymphocyte, neutrophil, and platelet count, as well as creatinine, alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) levels, and disease severity were analyzed. We found 8 different mtDNA mutations in ND1, ND5, CO3, ATP6, and CYB genes, which were predicted to alter amino acids and decrease protein stability. Two missense unique mutations, C9555T in CO3 and A12418T in ND5 were identified and correlated with Complexes I and IV, respectively. This downregulation was correlated with age, elevated levels of leukocytes, lymphocytes, neutrophils, platelets, creatinine, ALT, AST, and BUN, as well as disease severity. These findings suggest that mtDNA mutations and altered expression of oxidative phosphorylation genes contribute to mitochondrial dysfunction in COVID-19. Targeting mitochondrial dysfunction may represent a promising therapeutic strategy for COVID-19 treatment.