Non-cyanobacterial diazotrophs (NCDs) are widespread and active in marine waters. The carbon and low-oxygen (O2) conditions required for their N2 fixation may be encountered on marine particles, while a putative role of light remains uninvestigated. This study explored factors that regulate N2 fixation in Rhodopseudomonas sp. BAL398—a anoxygenic phototrophic bacterium isolated from low-salinity surface waters. Light (250 µmol photons m−2 s−1) and anoxia (0 µM O2) stimulated growth and N2 fixation; however, diazotrophy in light was dependent on high organic carbon levels (35 mM, glucose:succinate). Immunolabeling revealed that cellular nitrogenase levels increased with light, decreasing inorganic nitrogen (N) and ambient O2 (250 µM). Light and O2 stimulated motility and biofilm formation on surfaces, and N2 fixation rates increased compared to the control treatment. N2 fixation rates were positively correlated with the formation of rosette-like cellular structures, and an increased concentration of nitrogenase was observed toward the center of these structures, which increased their occurrence 600 times when cultures reached maximum N2 fixation rates vs when they had low rates. Interestingly, N2 fixation was not completely inhibited under oxic conditions and was accompanied by increased formation of capsules and cysts. Rosettes, as well as capsules and cysts, may thus serve as protection against O2. Our study reveals the physiological adaptations that underlie N2 fixation in an anoxygenic phototroph, emphasizing the significance of biofilm formation for utilizing light and fixing N2 under oxic conditions, and underscores the need for deciphering the importance of light for marine NCDs.