Multiple fluorochromes are extensively used to investigate different microalgal aspects, such as viability and physiology. Some of them can be used to stain nucleic acids (DNA). Well-known examples are SYBR Green I and SYTO 9, the latter of which offers several advantages, especially when combined with flow cytometry (FCM)—a powerful method for studying microalgal population heterogeneity and analyzing their cell cycles. However, the effects of these dyes on the microalgae cell physiology have not been fully elucidated yet. A statistical experimental design, using response surface methodology (RSM) with FCM was applied in this study to optimize the DNA staining of a non-conventional microalgae, Chromochloris zofingiensis, with SYBR Green I and SYTO 9, and to optimize the variables affecting staining efficiency, i.e., the dye concentration, incubation time and staining temperature. We found that none of these factors affects the staining efficiency, which was not less than 99.65%. However, for both dyes, the dye concentration was shown to be the most significant factor causing cell damage (p-values: 0.0003; <0.0001) for SYBR Green I and SYTO 9, respectively. The staining temperature was only significant for SYTO 9 (p-value: 0.0082), and no significant effect was observed regarding the incubation time for both dyes. The values of the optimized parameters (0.5 µM, 05 min and 25 °C) for SYTO 9 and (0.5 X, 5 min and 25 °C) for SYBR Green I resulted in the maximum staining efficiency (99.8%; 99.6%), and the minimum damaging effects (12.86%; 13.75%) for SYTO 9 and SYBR Green I, respectively. These results offer new perspectives for improving the use of DNA staining fluorochromes and provides insights into their possible side effects on microalgae.