Interactions between bacteria and eukaryotic phytoplankton have major influences on marine ecosystems and the global carbon cycle. Metabolite exchanges between specific bacteria and phytoplankton species result in beneficial or antagonistic associations that influence the physiology and productivity of both groups and occur within the microenvironment of the phytoplankton host, termed the phycosphere. Current sampling techniques to study these interactions are limited to sampling batch cultures or large-volume seawater, which obscures distinction between phycosphere associated bacteria and free-living bacteria. Single-cell isolation methods, such as fluorescence-activated cell sorting (FACS), have been used to isolate phytoplankton and bacteria; however, FACS only enables isolation of bacteria tightly associated with phytoplankton cells but not those loosely associated or ones that track the phycosphere using chemotaxis. Such techniques also are not amenable to large phytoplankton cell sizes or chains. Here, we present a step-by-step protocol for the isolation of phytoplankton cells and the bacteria within their phycosphere using the PhycoPick, a microosmotic pump manufactured to enable robust micro-isolation and transfer of small particles without pulsation. The PhycoPick is able to isolate volumes in the pico-to-nanoliter range, which is ideal for capturing bacteria in the phycosphere while avoiding capturing free-living bacteria. The phycospheres are carefully isolated through intuitive manipulation of the micropump inflow and outflow controls to enable the isolation of a single phytoplankton cell or chain along with the small volume of water surrounding it. These phycospheres can then be used for downstream analyses, including phytoplankton cultivation, bacterial enumeration and cultivation, amplicon and shotgun metagenomics sequencing of these communities, and targeted metabolomics of phycosphere metabolites using mass spectrometry. The PhycoPick is a powerful system to query the interactions between these microeukaryotic hosts and their bacteria in culture or microbial communities in aquatic environments.