Pre-targeting is a proven strategy for in vivo delivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which is based on copper-free "click" chemistry. Copper-free click reactions have shown in vivo potential for imaging and radionuclide therapy, but this conjugation strategy has not yet been explored in combination with microspheres or unicellular organisms. This study aims to evaluate the in vivo efficacy of strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to achieve imaging and targeting of azide-functionalized macro-aggregated albumin (MAA) microspheres and Staphylococcus aureus bacteria. MAA microspheres (diameter 10-90 μm) were functionalized with a biorthogonal Cy5 fluorophore, bearing an azide functionality (N3), to generate MAA-Cy5-N3. S. aureus (diameter ∼1 μm) were functionalized with 99mTc-UBI29-41-Cy5-N3, generating S. aureus-99mTc-UBI29-41-Cy5-N3. In situ and in vitro click conjugation on the -N3 moieties was studied for 20 h using a radioactivity-based assay and fluorescence microscopy. For in vivo validation, both primary entities, radiolabeled with 99mTc, were deposited into the microvasculature of the liver via intrasplenic injections. Secondary targeting was realized following the intravenous administration of indium-111-radiolabeled diethylenetriaminepentaacetic acid-dibenzocyclooctyne (111In-DTPA-DBCO). To assess click reaction efficiency in vivo, 99mTc and 111In-biodistributions were measured (SPECT and %ID g-1). Use of 111In-DTPA-DBCO in mice without MAA deposits or mice infected with non-functionalized S. aureus served as controls. Ex vivo confocal fluorescence imaging was carried out in excised tissues to confirm the presence of functionalized MAA and bacteria. In vitro data confirmed effective click reactions on both the MAA particles and the bacterial membrane. SPECT imaging and biodistribution studies revealed significantly (p < 0.05) increased accumulation of 111In-DTPA-DBCO at the sites where MAA-Cy5-N3 (7.5 ± 1.5%ID g-1vs. 3.5 ± 0.5%ID g-1 in control mice) and S. aureus-99mTc-UBI29-41-Cy5-N3 (9.3 ± 1.3%ID g-1vs. 6.0 ± 0.5%ID g-1 in control mice) resided. Ex vivo fluorescence imaging confirmed the presence of either functionalized MAA or S. aureus in excised spleens and livers of mice. Copper-free click chemistry between a DBCO moiety and Cy5-N3-functionalized microspheres or bacterial entities in the liver can be used to realize in vivo imaging and targeting.