Acetylcholine (ACh) release in the medial prefrontal cortex (mPFC) is a major contributor to the balance between attention and inhibitory control, which is crucial for the execution of adaptive goal-directed behaviors. Yet, our understanding of the functional heterogeneity within the mPFC, particularly how distinct cholinergic afferences and circuits regulate these processes, remains limited. Here, we used in vivo fiber photometry, neuronal tracing, and chemogenetics manipulations to demonstrate the role of the prelimbic sub-region of the mPFC (PrL) and its ascending cholinergic projections in a cued-Fixed Consecutive Number task (FCNcue task) in male mice. We found that following a transient activation at the initiation of the behavioral response (cue detection), persistent inhibition of PrL neuronal activity, measured by fiber photometry, may be necessary to maintain engagement in the task and completion of the chain of required responses (i.e., optimal responses). Moreover, we found that the PrL receives dense ACh projections almost exclusively from the most anterior-medial part of the basal forebrain (BF) comprising the horizontal and ventral parts of the diagonal band of Broca (HDB and VDB) and the substantia innominata (SI) nuclei. Finally, chemogenetic activation of this ACh pathway inhibited PrL activity and enhanced behavioral performance of the mice by increasing the percentage of optimal responses. Overall, this study provides insights into the spatial and temporal dynamics of cholinergic signaling to the PrL and its causal role on attentional control.