Spatial working memory (SWM) is the ability to encode, maintain, and retrieve spatial information over a temporal gap, and relies on a network of structures including the medial septum (MS), which provides critical input to the hippocampus. Although the role of the MS in SWM is well-established, up until recently, we have been unable to use temporally precise circuit manipulation techniques to examine the specific role of the MS in SWM, particularly to distinguish between encoding, maintenance, and retrieval. Here, we test the hypothesis that the MS supports the maintenance of spatial information over a temporal gap using precisely timed optogenetic suppression delivered during specific portions of three different tasks, two of which rely on SWM and one that does not. In experiment 1, we found that MS optogenetic suppression impaired choice accuracy of a SWM dependent conditional discrimination task. Moreover, this deficit was only observed when MS suppression was delivered during the cue-sampling, but not the cue-retrieval, portion of the trial. There was also no deficit when MS neurons were optogenetically suppressed as rats performed a SWM-independent variant of the task. In experiment 2, we tested whether MS suppression affected choice accuracy on a delayed nonmatch to position (DNMP) task when suppression was limited to the sample, delay, and choice phases of the task. We found that MS suppression delivery during the delay phase of the DNMP task, but not during the sample or choice phases, impaired choice accuracy. Our results collectively suggest that the MS plays an important role in SWM by maintaining task-relevant information over a temporal delay.