Numerous studies have reported the beneficial effects of acute exercise on executive functions. Less is known, however, about the effects of exercise on working memory as one subcomponent of executive functions and about its effects on older adults. We investigated the effects of acute moderate-intensity exercise on working memory performance, the respective cortical hemodynamic activation patterns, and the development and persistence of such effects in healthy older adults. Forty-four participants (: 69.18 years ± 3.92; 21 females) performed a letter 2-back task before and at three time points after (post 15 min, post 30 min, and post 45 min) either listening to an audiobook or exercising (15 min; 50% VO-peak). Functional near-infrared spectroscopy (fNIRS) was used to assess cortical hemodynamic activation and brain-behavior correlations in the fronto-parietal working memory network. Overall, we found no group differences for working memory performance. However, only within the experimental group, 2-back performance was enhanced 15 min and 45 min post-exercise. Furthermore, 15 min post-exercise frontal activation predicted working memory performance, regardless of group. In sum, our results indicate slight beneficial effects of acute moderate-intensity exercise on working memory performance in healthy older adults. Findings are discussed in light of the cognitive aging process and moderators affecting the exercise-cognition relationship.