This study aimed to clarify whether short-term neurofeedback training during the acute stroke phase led to prefrontal activity self-regulation, providing positive efficacy to working memory. A total of 30 patients with acute stroke performed functional near-infrared spectroscopy-based neurofeedback training for a day to increase their prefrontal activity. A randomized, Sham-controlled, double-blind study protocol was used comparing working memory ability before and after neurofeedback training. Working memory was evaluated using a target-searching task requiring spatial information retention. A decline in spatial working memory performance post-intervention was prevented in patients who displayed a higher task-related right prefrontal activity during neurofeedback training compared with the baseline. Neurofeedback training efficacy was not associated with the patient’s clinical background such as Fugl–Meyer Assessment score and time since stroke. These findings demonstrated that even short-term neurofeedback training can strengthen prefrontal activity and help maintain cognitive ability in acute stroke patients, at least immediately after training. However, further studies investigating the influence of individual patient clinical background, especially cognitive impairment, on neurofeedback training is needed. Current findings provide an encouraging option for clinicians to design neurorehabilitation programs, including neurofeedback protocols, for acute stroke patients.