This dataset comprises measurements of microbial uptake activities of betaine and choline, particulate phase osmolytes, amplicon sequencing of marker genese involved in Nitrogenous-osmolyte catabolism, and single cell genome data. Water samples were collected from at the L4 station of the Western Channel Observatory between April 27, 2015 to April 24, 2017 using Niskin bottles attached to a rosette sampler deployed from the RV Plymouth Quest. Nitrogenous osmolytes (glycine betaine, choline and trimethylamine N-oxide are essential components for most organisms in the marine environment. They enable cells to exist in a salty environment, but also have several other proposed uses. The aim of the project is to understand the seasonal cycle of glycine betaine, trimethylamine N-oxide and choline at Station L4. The water samples were analysed for the microbial assimilation and dissimilation activities using 14C labelled betaine and choline, respectively. The data will be incorporated to the European Regional Seas Ecosystem Model (ERSEM) coupled with the hydrodynamic model General Ocean Turbulent Model (GOTM) to simulate the N-osmolyte cycling at the L4 station. The data were collected under the project Biogeochemical cycling of N-osmolytes in the surface ocean funded by NERC Discovery Science grants NE/M002233/1 (parent), NE/M003361/1 (child), NE/M002934/1 (child). The grants were led by Dr Yin Chen, Dr Ruth Airs, and Dr Wei Huang respectively.