PLX5622 is a small molecular inhibitor of the CSF1 receptor (CSF1R) and is widely used to deplete macrophages within the central nervous system (CNS). However, recent reports have indicated that PLX5622 may affect myeloid cells in other organs including the bone marrow and spleen. We investigated the impact of PLX5622 treatment in wild-type C57BL/6 mice and discovered that one-week treatment with PLX5622 was sufficient to deplete interstitial macrophages in the lung and brain-infiltrating Ly6Clow patrolling monocytes, in addition to CNS-resident macrophages. These cell types were previously indicated to act as infection reservoirs for the pathogenic fungus Cryptococcus neoformans. We therefore took advantage of PLX5622-mediated depletion of these myeloid cell subsets to examine their functional role in C. neoformans lung infection and extrapulmonary dissemination. We found that PLX5622-treated mice had significantly reduced fungal lung infection and reduced extrapulmonary dissemination to the CNS but not to the spleen or liver. Fungal lung infection mapped to MHCIIhi interstitial lung macrophages, which underwent significant expansion during infection following monocyte replenishment and not local division. Although PLX5622 depleted CNS infiltrating patrolling monocytes, these cells did not accumulate in the fungal-infected CNS following pulmonary infection. In addition, Nr4a1-deficient mice, which lack patrolling monocytes, had similar control and dissemination of C. neoformans infection to wild-type controls. Our data demonstrate that PLX5622 may have a beneficial effect in the control of intracellular replicating pathogenic fungi that utilise CSF1R-dependent myeloid cells as infection reservoirs.