Catharanthus roseus L. is a valuable medicinal plant renowned for its potent anticancer compounds, i.e., vincristine and vinblastine. The efficient mass production of this plant can be challenging due to limitations in conventional propagation methods. Tissue culture techniques offer a promising alternative, and optimizing plant growth regulator (PGR) treatments can play a crucial role in achieving successful in vitro propagation. In this study, we investigated the effects of several PGR combinations, i.e., NAA, BAP, and 2,4-D, on the morpho-phytochemical attributes and antioxidant activity of in vitro grown Catharanthus roseus L. var. ‘Ocellata’. The plants were arranged in a completely randomized design with three replications. The results demonstrated that applying PGRs, either alone or in combination, significantly improved the morphological characteristics. The NAA treatment caused the highest plant height, number of branches and leaves, and root diameter. Notably, the treatment with 2,4-D resulted in the highest photosynthetic pigment content compared to the control. Moreover, the treatments with NAA + BAP and NAA + BAP + 2,4-D exhibited the highest levels of total phenolic compounds (TPC) and total flavonoid content (TFC) in both roots and leaves. Regarding antioxidant activity, the DPPH radical scavenging assay revealed the highest radical scavenging percentages, i.e., 74.21% (NAA + 2,4-D) in leaf samples and 78.08% (NAA + 2,4-D) in root samples. These findings emphasize the potency of PGRs in optimizing tissue culture protocols for Catharanthus roseus L., thus facilitating the production of superior-quality plants with enriched medicinal properties. This study contributes to the advancement of sustainable and efficient cultivation of bioactive compounds from Catharanthus roseus L. using tissue culture techniques.