Burkholderia mallei and B. pseudomallei cause glanders and melioidosis, respectively, in humans and animals. A hallmark of pathogenesis is the formation of granulomas containing multinucleated giant cells (MNGCs) and cell death. These processes depend on type 6 secretion system 1 (T6SS-1), which is required for virulence in animals. We examined the cell biology of MNGC formation and cell death. We found that chloroquine diphosphate (CLQ), an antimalarial drug, inhibits Burkholderia growth, phagosomal escape, and subsequent MNGC formation. This depends on CLQ's ability to neutralize the acid pH because other alkalinizing compounds similarly inhibit escape and MNGC formation. CLQ inhibits bacterial virulence protein expression because T6SS-1 and some effectors of type 3 secretion system 3 (T3SS-3), which is also required for virulence, are expressed at acid pH. We show that acid pH upregulates the expression of Hcp1 of T6SS-1 and TssM, a protein coregulated with T6SS-1. Finally, we demonstrate that CLQ treatment of Burkholderia-infected Madagascar hissing cockroaches (HCs) increases their survival. This study highlights the multiple mechanisms by which CLQ inhibits growth and virulence and suggests that CLQ be further tested and considered, in conjunction with antibiotic use, for the treatment of diseases caused by Burkholderia.