Recognition-based assembly of micron- to nano-sized colloidal particles functionalized with DNA has generated great interest in the past decade; however, reversing the assembly process is typically achieved by thermal denaturation of the oligonucleotide duplexes. Here, we report an alternative disassembly approach at a fixed temperature using competitive hybridization events between immobilized and soluble oligonucleotide strands. Microspheres are first aggregated via primary hybridization events between immobilized DNA strands with a weak, but sufficient, affinity for partner strands to link complementary surfaces together. To reverse the aggregation process, soluble oligonucleotides are then added to competitively displace the original hybridization partners through secondary hybridization events. Using flow cytometry to quantify hybridization events and microscopy to examine DNA-mediated aggregation and redispersion, we found that the efficiency of competitive displacement is based upon (1) the difference in base pair matches between the primary and secondary target for the same probe sequence and (2) the concentration of hybridizing oligonucleotides participating in microsphere aggregation. To the best of our knowledge, this study is the first to employ DNA hybridization events to mediate reversible adhesion between colloidal particles at a fixed temperature.