Microalgae and diatoms, as microscopic unicellular algae, present a promising alternative for biodiesel production due to their high lipid content and rapid growth rates. This study aimed to evaluate the effects of varying salinity levels and different concentrations of silicate and nitrate on the biomass and lipid production of the marine diatom Chaetoceros muellerii OQ390046. Cultures were grown in f/2 media enriched with NaCl at concentrations of 15 ppt, 30 ppt, 45 ppt, and 60 ppt, and with Na2SiO3 at 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM, along with NaNO3 at concentrations of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, and 30 mM, and KH2PO4 at 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, and 30 mM. The cultures were maintained under controlled conditions in a plant growth chamber. The results indicated that, under varying salinity conditions, the highest biomass production (6.7 g/L) was achieved at 45 ppt salinity, while the maximum lipid content (3.2 mg/L) was observed at 60 ppt. Among the tested silicate concentrations, the highest biomass (5.7 g/L) was recorded with 0.05 mM Na2SiO3, and the highest lipid production (10.4 mg/L) was noted with 0.02 mM Na2SiO3. Furthermore, the study revealed that light intensity also played a crucial role in maximizing production; 1000 lux was optimal for biomass accumulation, whereas 100 lux was most effective for enhancing lipid production. These findings highlight the potential for optimizing culture conditions to improve both biomass and lipid yields in Chaetoceros muellerii, thus advancing its application in biodiesel production.