Background: Many histone deacetylase (HDAC) inhibitors have demonstrated preclinical efficacy as monotherapy or in combination with other anticancer drugs for both hematological and solid tumor malignancies. In clinical, however, the HDAC inhibitors have proven less effective as single agents. Thus, prompting the investigation of rational combination of HDAC inhibitors with other therapeutics. Objectives: This study aims to evaluate the potential of Macrotyloma uniflorum in facilitating the potency of histone deacetylase inhibitor, Vorinostat-suberoylanilide hydroxamic acid (SAHA). Materials and Methods: Powdered seeds of M. uniflorum were sequentially extracted with water, methanol, ethyl acetate, and hexane. The quantitative analysis of the different extracts was carried out. The cytotoxicity of M. uniflorum seed extracts was investigated on the leukemia cell line (HL-60). HeLa cells were used to study the histone deacetylase inhibitory activity of the extracts. Further, the effect of the combination of M. uniflorum seed extract and SAHA was studied. Results: The aqueous extract showed the highest amount of both phenolic (311.21 ± 2 mg gallic acid/g) and flavonoid content (89.96 ± 4.62 mg Quercetin/g extract). The combination showed more specificity toward class I HDAC isoforms among the different isoforms (class I, class IIa, class IIb, and class IV). Moreover, M. uniflorum alone showed no significant cytotoxicity on the HL-60 cell line. However, in combination with SAHA, the aqueous extract exhibited significant cytotoxic activity. Further, the results show that the extract of M. uniflorum has a significant effect on cell death and autophagy in leukemia. Conclusion: Mechanistic studies revealed that aqueous extract along with SAHA facilitated HDAC inhibitory activity, cell death, and autophagy.