Two nuclear genes, ACTN3, encoding for the α-actinin skeletal muscle isoform 3, and ACE encoding the angiotensin-converting enzyme, have both been associated with quantitative physical performance traits in the general population. The purpose of our study was to assess the association between the two nuclear gene variants, R577X (rs1815739) in ACTN3 and I/D (rs4340) in ACE, with elite athletes performance and the effect of training on the mitochondrial DNA (mtDNA) content in peripheral blood. We evaluated the genotypes and frequencies of ACTN3 R577X and ACE I/D polymorphisms between soccer players (n = 43) and healthy non-athletic controls (n = 128). Total DNA was extracted from peripheral blood samples using the standard procedure. The genotypes were assessed by PCR-RFLP analysis and mtDNA cellular content by RT-PCR. The soccer players showed a tendency to a prevalence of ACTN3RR and ACEDD genotypes both independently and in co-occurrence. The effect of physical training on the mitochondrial DNA content in the athletic population was reflected strikingly in its increase in peripheral blood. Based on our results, we suggest that the analysis of ACTN3 and ACE genotypes could predict talent in the soccer field and that knowledge of the genetic variants could determine types and training times for soccer players. In addition, the novelty of this work, never before described in the sports literature, is that the increase of mitochondrial content can be correlated with the training load, suggesting that the mtDNA copy number may be considered a viable bioenergetics biomarker.