Abstract High-sensitivity C-reactive protein (hsCRP) and homocysteine (Hcy) are inflammation markers but are also related to cardiovascular diseases, disability, or higher risk of death. Although inflammation is considered to be associated with frailty, data regarding the association between hsCRP or Hcy and frailty are controversial or scarce, especially with respect to their association with prefrailty. Thus, our objective was to study the association of hsCRP and Hcy with prefrailty and frailty in 1,211 Spanish men and women aged 65–98 years from the Toledo Study for Healthy Aging (TSHA) cohort, classified according to Fried’s criteria. Hcy was independently associated with frailty (odds ratio [OR] = 1.06; 95% confidence interval [CI]: 1.01–1.12), whereas hsCRP was independently associated with both prefrailty (OR = 1.03; 95% CI: 1.01–1.06) and frailty (OR = 1.07; 95% CI: 1.02–1.12). Furthermore, both markers were positively correlated with the number of Fried’s criteria that were met and were independently associated with the criteria of exhaustion (Hcy: OR = 1.03, 95% CI: 1.00–1.06), weakness (hsCRP: OR = 1.03, 95% CI: 1.01–1.05), and low physical activity (hsCRP: OR = 1.04, 95% CI: 1.02–1.06). Thus, our results highlight the importance of inflammation in age-related physical decline and, in particular, its association with fatigue, low strength, and decreased physical activity.