B vitamins are essential cofactors for practically all living organisms on Earth and are produced by a selection of microorganisms. An imbalance between high demand and limited production, in concert with abiotic processes, may explain the low availability of these vitamins in marine systems. Natural microbial communities from surface shelf water in the productive area off northwestern Spain were enclosed in mesocosms in winter, spring, and summer 2016. In order to explore the impact of B-vitamin availability on microbial community composition (16S and 18S rRNA gene sequence analysis) and bacterial function (metatranscriptomics analysis) in different seasons, enrichment experiments were conducted with seawater from the mesocosms. Our findings revealed that significant increases in phytoplankton or prokaryote biomass associated with vitamin B-12 and/or B-1 amendments were not accompanied by significant changes in community composition, suggesting that most of the microbial taxa benefited from the external B-vitamin supply. Metatranscriptome analysis suggested that many bacteria were potential consumers of vitamins B-12 and B-1, although the relative abundance of reads related to synthesis was ca. 3.6-fold higher than that related to uptake. Alteromonadales and Oceanospirillales accounted for important portions of vitamin B-1 and B-12 synthesis gene transcription, despite accounting for only minor portions of the bacterial community. Flavobacteriales appeared to be involved mostly in vitamin B-12 and B-1 uptake, and Pelagibacterales expressed genes involved in vitamin B-1 uptake. Interestingly, the relative expression of vitamin B-12 and B-1 synthesis genes among bacteria strongly increased upon inorganic nutrient amendment. Collectively, these findings suggest that upwelling events intermittently occurring during spring and summer in productive ecosystems may ensure an adequate production of these cofactors to sustain high levels of phytoplankton growth and biomass. IMPORTANCE B vitamins are essential growth factors for practically all living organisms on Earth that are produced by a selection of microorganisms. An imbalance between high demand and limited production may explain the low concentration of these compounds in marine systems. In order to explore the impact of B-vitamin availability on bacteria and algae in the coastal waters off northwestern Spain, six experiments were conducted with natural surface water enclosed in winter, spring, and summer. Our findings revealed that increases in phytoplankton or bacterial growth associated with B-12 and/or B-1 amendments were not accompanied by significant changes in community composition, suggesting that most microorganisms benefited from the B-vitamin supply. Our analyses confirmed the role of many bacteria as consumers of vitamins B-12 and B-1, although the relative abundance of genes related to synthesis was ca. 3.6-fold higher than that related to uptake. Interestingly, prokaryote expression of B-12 and B-1 synthesis genes strongly increased when inorganic nutrients were added. Collectively, these findings suggest that upwelling of cold and nutrient-rich waters occurring during spring and summer in this coastal area may ensure an adequate production of B vitamins to sustain high levels of algae growth and biomass.