The resistance of fungal pathogens to traditional antifungal medications presents a significant public health concern, particularly for immunocompromised patients, leading to elevated rates of illness and death. This study aimed to explore the antifungal properties of zinc oxide nanoparticles (ZnO NPs) produced using a natural approach involving the aqueous seed extract of Trigonella foenumgracium (Fenugreek). Additionally, the study assessed the combined effectiveness of these biologically synthesized ZnO NPs with fluconazole against fungal pathogens. The biologically synthesized ZnO NPs were observed to have a hexagonal shape with an average diameter of 27 nm and a surface charge of -18.3 mV. Notably, these nanoparticles exhibited the most potent antifungal activity against Candida tropicalis, with a relative inhibition zone diameter of 18.67±0.56 mm. The minimum inhibitory concentration against C. tropicalis was determined to be 100 μg/mL, while the minimum fungicidal concentration was found to be 200 μg/mL. The greatest synergistic effect between the biologically synthesized ZnO NPs and fluconazole was observed against Candida glabrata, followed by Candida parapsilosis. In summary, the study underscores the potential of combining biologically synthesized ZnO NPs with fluconazole to formulate an effective antifungal treatment against drug-resistant fungal pathogens, thereby enhancing the therapeutic efficacy of conventional antifungal drugs.