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Objective Positive psychological constructs, such as optimism, are associated with cardiovascular health, and changes in biological measures associated with heart health have been proposed as potential mediators of these relationships. In this analysis of data from a randomized controlled trial, we examined the impact of an optimism training intervention on biological measures associated with cardiac health in patients with coronary artery disease. Methods We analyzed data from an 8-week, randomized, controlled trial of a group-based optimism training intervention in 61 patients with coronary artery disease. High-sensitivity C-reactive protein (hs-CRP), interleukin 6, irisin, and fibrinogen were measured at baseline, 8 weeks, and 16 weeks. Mixed-effects regression analyses were performed to examine the effects of the intervention on changes in biological measures at 8 and 16 weeks. Results At 8 weeks, the intervention led to significantly greater reductions in hs-CRP (B = −0.851 [standard error {SE} = 0.273, p = .002) and fibrinogen (B = −0.148 [SE = 0.062], p = .016), and a greater increase in irisin (B = 0.252 [SE = 0.114], p = .027) compared with the control condition. These changes persisted at 16 weeks (hs-CRP: B = −1.078 [SE = 0.276], p < .001; fibrinogen: B = −0.270 [SE = 0.062], p < .001; irisin: B = 0.525 [SE = 0.116], p < .001), and interleukin 6 additionally was impacted at this time point (B = −0.214 [SE = 0.064], p = .001). Exploratory mediation analyses failed to identify significant psychological or health behavior mediators of these relationships. Conclusions A group-based optimism training intervention resulted in significant, robust, and sustained changes in biological measures associated with cardiac health. Further studies are needed to confirm these findings in a larger sample and identify potential mediating variables. Trial Registration: Iran Registry of Clinical Trials No. 2016070328769 N1.