A novel instrument to manipulate and characterize the mechanical environment in and around microscale objects in a fluidic environment has been developed by integrating two laser-based techniques: micron-resolution particle image velocimetry (µPIV) and optical tweezers (OT). This instrument, the µPIVOT, enables a new realm of microscale studies, yet still maintains the individual capabilities of each optical technique. This was demonstrated with individual measurements of optical trap stiffness (~70 pN µm⁻¹ for a 20 µm polystyrene sphere and a linear relationship between trap stiffness and laser power) and fluid velocities within 436 nm of a microchannel wall. The integrated device was validated by comparing computational flow predictions to the measured velocity profile around a trapped particle in either a uniform flow or an imposed, gravity-driven microchannel flow (R² = 0.988, RMS error = 13.04 µm s⁻¹). Interaction between both techniques is shown to be negligible for 15 µm to 35 µm diameter trapped particles subjected to fluid velocities from 50 µm s⁻¹ to 500 µm s⁻¹ even at the highest laser power (1.45 W). The integrated techniques will provide a unique perspective toward understanding microscale phenomena including single-cell biomechanics, non-Newtonian fluid mechanics and single particle or particle–particle hydrodynamics.