The biosynthesis of metallic nanoparticles (NPs) has been previously reported using a variety of organic molecules produced by microalgae. However, the results obtained could vary due to the metabolic responses that microalgae have to different culture conditions which could affect the characteristics of the produced nanoparticles. In the present report, copper nanoparticle formation was evaluated by the microalgae Chlorella kessleri, Dunaliella tertiolecta, and Tetraselmis suecica, developed under combined conditions of low (L−) and high (L+) illumination, with low (S−) and high salinity (S+). The illumination was 12 h:12 h light/dark. NP formation was evaluated 72 h after exposure to copper salt. Cupric oxide (CuO) NPs were detected spectrophotometrically in both the culture media (extracellular NPs) and cells (intracellular NPs) of Ch. kessleri with absorbance in the range of 200 to 235 nm. Metallic copper NPs (Cun) were detected with an absorbance between 540 and 560 nm in treatments with cells of C. kessleri and D. tertiolecta which were grown in L+S−, while T. suecica cells showed Cun NPs formations in L−S−, L−S+, and L+S−. The size difference of the NPs was measured by scanning electron microscopy (SEM), in treatments with cells of C. kessleri, ranging in size from 15 to 25 nm (L−S−) and 55 to 65 nm (L+S−). In treatments with culture media, sizes from 35 to 45 nm (L−S−) of NPs were obtained. Differences in the biosynthesis of Cu-based NPs are possible, depending on the culture conditions and the strain of microalgae to be utilized.