Marine phytoplankton have conserved elemental stoichiometry, but there can be significant deviations from this Redfield ratio. Moreover, phytoplankton allocate reduced carbon (C) to different biochemical pools based on nutritional status and light availability, adding complexity to this relationship. This allocation influences physiology, ecology, and biogeochemistry. Here, we present results on the physiological and biochemical properties of two evolutionarily distinct model marine phytoplankton, a diatom (cf. Staurosira sp. Ehrenberg) and a chlorophyte (Chlorella sp. M. Beijerinck) grown under light and nitrogen resource gradients to characterize how carbon is allocated under different energy and substrate conditions. We found that nitrogen (N)-replete growth rate increased monotonically with light until it reached a threshold intensity (~200 μmol photons · m−2 · s−1). For Chlorella sp., the nitrogen quota (pg · μm−3) was greatest below this threshold, beyond which it was reduced by the effect of N-stress, while for Staurosira sp. there was no trend. Both species maintained constant maximum quantum yield of photosynthesis (mol C · mol photons−1) over the range of light and N-gradients studied (although each species used different photophysiological strategies). In both species, C:chl a (g · g−1) increased as a function of light and N-stress, while C:N (mol · mol−1) and relative neutral lipid:C (rel. lipid · g−1) were most strongly influenced by N-stress above the threshold light intensity. These results demonstrated that the interaction of substrate (N-availability) and energy gradients influenced C-allocation, and that general patterns of biochemical responses may be conserved among phytoplankton; they provided a framework for predicting phytoplankton biochemical composition in ecological, biogeochemical, or biotechnological applications.