Withania coagulans contains a complex mixture of various bioactive compounds. In order to reduce the complexity of the plant extract to purify its phytochemical biomolecules, a novel fractionation strategy using different solvent combination ratios was applied to isolate twelve bioactive fractions. These fractions were tested for activity in the biogenic synthesis of cobalt oxide nanoparticles, biofilm and antifungal activities. The results revealed that plant extract with bioactive fractions in 30% ratio for all solvent combinations showed more potent bioreducing power, according to the observed color changes and the appearance of representative absorption peaks at 500–510 nm in the UV-visible spectra which confirm the synthesis of cobalt oxide nanoparticles (Co₃O₄ NPs). XRD diffraction was used to define the crystal structure, size and phase composition of the products. The fractions obtained using 90% methanol/hexane and 30% methanol/hexane showed more effectiveness against biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus so these fractions could potentially be used to treat bacterial infections. The 90% hexane/H₂O fraction showed excellent antifungal activity against Aspergillus niger and Candida albicans, while the 70% methanol/hexane fraction showed good antifungal activity for C. albicans, so these fractions are potentially useful for the treatment of various fungal infections. On the whole it was concluded that fractionation based on effective combinations of methanol/hexane was useful to investigate and study bioactive compounds, and the active compounds from these fractions may be further purified and tested in various clinical trials.