As longevity is increasing, the 65-year-old and older population is projected to increase in the next decades, as are the consequences of age-related muscle deterioration on the quality of life. The purpose of this study was to examine the associations of the ACTN3R577X polymorphism with quality of life and muscular strength in an older Spanish population. In total, 281 older adults participated in this study. Anthropometric measurements, chronic diseases, prescribed medications, quality of life, hand grip strength, and physical activity and nutritional status data were collected. ACTN3 R577X genotyping was determined using Taqman probes. Multivariate regression analysis revealed in adjusted model that, in men, the ACTN3 R577X genotype was significantly associated with hand grip strength (HGS), regression coefficient (β) = 1.23, p = 0.008, dimension 1 of the five-dimension questionnaire EuroQoL (EQ-5D, mobility), (β) = −1.44, p = 0.006, and clinical group risk (CGR) category (β) = −1.38, p = 0.006. In women, a marginal association between the ACTN3 R577X genotype and the CGR category was observed, with a regression coefficient of (β) = −0.97, (p = 0.024). Our findings suggest that the ACTN3 R577X genotype may influence the decline in muscle strength and quality of life in older Spanish adult males.