Microplastics (MPs) have become pervasive in marine ecosystems, potentially causing environmental degradation, impacting ecological function, and posing a serious public health risk. Despite the widespread distribution of MPs, their vertical transport within a water column has limited understanding, representing a key knowledge gap in the development of water quality models to minimize these risks. In this study, 6152 individual particles of six common types of MPs were observed through water column experiments to examine a range of drivers of the vertical velocity of MPs, including particle density and size, biofilm growth, water temperature, and salinity. The experimental results revealed that the vertical velocity of MPs obeyed Stokes’ law under laminar conditions; increasing salinity decreased the settling tendency of the particles. Moreover, biofilm attachment induced notable alterations in particle characteristics within 60 days, resulting in slower settling velocities (up to a 21.9% change for non-buoyant MPs) and even a reversed vertical direction (up to several times for buoyant particles). Furthermore, a lattice Boltzmann model could predict the vertical velocity of MPs with reasonable accuracy, especially for small particles. This work facilitates the development of sophisticated models/formulas that integrate particle morphology, hydrodynamics, and biological factors to enhance the understanding of MP transport through the river-to-coastal continuum.