The suitability of applying empirical conversion factors (eCFs) to determine bacterial biomass production remains unclear because seawater cultures are usually overtaken by phylotypes that are not abundant in situ. While eCFs vary across environments, it has not been tested whether differences in eCFs are driven by changes in bacterial community composition or by in situ environmental conditions. We carried out seawater cultures throughout a year to analyse the correlation between eCFs and bacterial community structure, analysed by catalysed reporter deposition fluorescence in situ hybridization. Gammaproteobacteria usually dominated seawater cultures, but their abundance exhibited a wide range (25–73% of cell counts) and significantly increased with inorganic nutrient enrichment. Flavobacteria were less abundant but increased up to 40% of cells counts in winter seawater cultures, when in situ chlorophyll a was high. The correlations between eCFs and the abundance of the main broad phylogenetic groups (Gamma-, Alphaproteobacteria and Flavobacteria) were significant, albeit weak, while more specific groups (Alteromonadaceae and Rhodobacteraceae) were not significantly correlated. Our results show that the frequent development of the fast-growing group Alteromonadaceae in seawater cultures does not strongly drive the observed variations in eCFs. Rather, the results imply that environmental conditions and the growth of specific phylotypes interact to determine eCFs.