Anterior alpha asymmetry is an EEG measure of relative left- or right-sided prefrontal cortical activity that has been associated with affective style, such that greater relative left-sided activity is associated with positive affect and approach-related behaviours. While mindfulness has been shown to enhance attention and affect, here we investigate the underlying neurobiological changes supporting these outcomes by assessing anterior alpha asymmetry. A longitudinal RCT was conducted to investigate the effect of an 8-week mindfulness training (MT) intervention on anterior alpha asymmetry and affect in a group of healthy adults aged over 60 years (n = 41). An active control computer-based attention training (CT) program (n = 26) designed to activate similar attentional components to mindfulness was used to determine if outcomes resulted from attention training or mindfulness-specific factors. The Sustained Attention to Response Task was used to assess attentional performance, while a breath counting task was used to classify mindfulness participants into high (MT-HIGH; n = 19) and low (MT-LOW; n = 22) proficiency groups. While all groups displayed improved attentional performance, only the MT-HIGH and MT-LOW groups showed significant increases in positive affect as measured by the Positive and Negative Affect Schedule. The MT-HIGH group showed significantly increased relative left-sided activity at both Fp1/Fp2 and F3/F4 electrode pairs, while no significant changes were observed in the MT-LOW and CT groups. These results suggest that 8 weeks of mindfulness training is capable of inducing changes in resting anterior alpha asymmetry, but these results are dependent upon the level of proficiency achieved.