This study combined measurements from multiple platforms with acoustic instruments on moorings and on a ship and optics on a profiler and an autonomous underwater vehicle (AUV) to examine the relationships between fluorescent, bioluminescent, and acoustically scattering layers in Monterey Bay during nighttime hours in July and August of 2006 and May of 2008. We identified thin bioluminescent layers that were strongly correlated with acoustic scattering at the same depth but were part of vertically broad acoustic features, suggesting layers of unique composition inside larger biomass features. These compositional thin layers nested inside larger biomass features may be a common ecosystem component and are likely to have significant ecological impacts but are extremely difficult to identify as most approaches capable of the vertical scales of measurement necessary for the identification of sub-meter scale patterns assess bulk properties rather than specific layer composition. Measurements of multiple types of thin layers showed that the depth offset between thin phytoplankton and zooplankton layers was highly variable with some layers found at the same depth but others found up to 16 m apart. The vertical offset between phytoplankton and zooplankton thin layers was strongly predicted by the fraction of the water column fluorescence contained within a thin phytoplankton layer. Thin zooplankton layers were only vertically associated with thin phytoplankton layers when the phytoplankton in a layer accounted for more than about 18–20% of the water column chlorophyll. Trophic interactions were likely occurring between phytoplankton and zooplankton thin layers but phytoplankton thin layers were exploited by zooplankton only when they represented a large fraction of the available phytoplankton, suggesting zooplankton have some knowledge of the available food over the entire water column. The horizontal extent of phytoplankton layers, discussed in the second paper in this series, is likely an important factor contributing to this selective exploitation by zooplankton. The pattern of vertical offset between phytoplankton and zooplankton layers was consistent between studies in different years and using different combinations of platforms, indicating the importance of the relationship between zooplankton layers and the fraction of phytoplankton within a layer at night within Monterey Bay. These results highlight the value of integrating measurements of various types of organisms to understand thin layers processes and the importance of assessing ecological interactions in plankton thin layers within the context of the properties of the entire water column, like the animals themselves do.