Diospyros villosa is traditionally used for an anti-bacterial property. Its cytotoxic effects have not been studied before. Therefore, this study aimed to examine the nutritional properties as well the cytotoxic effects of D. villosa. The leaves and stem barks were subjected to three different extraction methods (methanol, chloroform and hexane) and their nanoparticles were synthesized at two different temperatures (room temperature and at 80 °C). Thereafter, extracts were assessed using the associated AOCC protocols, for their nutritional content (moisture, fibre, proteins, lipid, ash and hydrolysable carbohydrates). Diospyros villosa extracts and their corresponding nanoparticles were then incubated overnight with cancerous and noncancerous cell lines to evaluate their cytotoxic potential. The nutritional analysis revealed that both young and mature leaves were rich sources of protein having values of 14.95% and 11.37% respectively. The moisture content was observed to be higher in all the leaf types (8.54 ± 0.75%, 9.67 ± 0.98% and 7.40 ± 0.80%) compared to the stem (2.13 ± 0.07%) respectively. The MTT cytotoxicity assay showed that the cell viability of MCF-7 cell lines was significantly lower when exposed to hexane and chloroform leaves extracts of D. villosa (IC50 of 26.64 and 26.07 µg mL−1) respectively, compared to camptothecin (36.54 µg mL−1). Similarly, the MCF-7 cell viability was observed to be significantly lower when exposed to hexane and chloroform stem extracts of D. villosa (IC50 of 24.57 and 3.92 µg mL−1), compared to camptothecin (36.54 µg mL−1). The cell viability of A549 cell lines was also found lower when exposed to the hexane and chloroform extracts (IC50 of 7.76 and 4.59 µg mL−1) compared to camptothecin (IC50 of 19.26 µg mL−1). Furthermore, the viability of A549 cell lines was found lower when exposed to hexane and chloroform stem extracts of D. villosa (IC50 of 10.67 and 5.35 µg mL−1) compared to camptothecin (19.26 µg mL−1). The biosynthesized nanoparticles further displayed an anticancer activity with an IC50 value of 4.08 µg mL−1 when compared to the control (36.54 µg mL−1). However, the HEK293 cell viability was observed to be significantly higher on exposure to hexane stem extracts of D. villosa (IC50 of 158.5 µg mL−1) compared to camptothecin (IC50 of 14.77 µg mL−1). Therefore, Diospyros villosa leaves, stem bark and nanoparticles synthesized showed high potential for being considered as a candidate for an anti-cancer regimen.