Nanoparticles derived from biological sources are currently garnering significant interest due to their diverse range of potential applications. The purpose of the study was to synthesize Al-doped nanoparticles of zinc oxide (ZnO) from leaf extracts of Cucumis maderaspatanus and assess their antioxidant and antimicrobial activity using some bacterial and fungal strains. These nanoparticles were analyzed using X-ray diffraction (XRD), ultraviolet–visible (UV-vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDAX), transmission electron microscopy (TEM), and thermogravimetric analysis/differential thermal analysis (TG-DTA). The average crystalline size was determined to be 25 nm, as evidenced by the XRD analysis. In the UV-vis spectrum, the absorption band was observed around 351 nm. It was discovered that the Al-ZnO nanoparticles had a bandgap of 3.25 eV using the Tauc relation. Furthermore, by FTIR measurement, the presence of the OH group, C=C bending of the alkene group, and C=O stretching was confirmed. The SEM analysis revealed that the nanoparticles were distributed uniformly throughout the sample. The EDAX spectrum clearly confirmed the presence of Zn, Al, and O elements in the Al-ZnO nanoparticles. The TEM results also indicated that the green synthesized Al-ZnO nanoparticles displayed hexagonal shapes with an average size of 25 nm. The doping of aluminum may enhance the thermal stability of the ZnO by altering the crystal structure or phase composition. The observed changes in TG, DTA, and DTG curves reflect the impact of aluminum doping on the structural and thermal properties of ZnO nanoparticles. The antibacterial activity of the Al-ZnO nanoparticles using the agar diffusion method showed that the maximum zone of inhibition has been noticed against organisms of Gram-positive S. aureus compared with Gram-negative E. coli. Moreover, antifungal activity using the agar cup method showed that the maximum zone of inhibition was observed on Aspergilus flavus, followed by Candida albicans. Al-doping nanoparticles increases the number of charge carriers, which can enhance the generation of reactive oxygen species (ROS) under UV light exposure. These ROS are known to possess strong antimicrobial properties. Al-doping can improve the crystallinity of ZnO, resulting in a larger surface area that facilitates more interaction with microbial cells. The structural and biological characteristics of Al-ZnO nanoparticles might be responsible for the enhanced antibacterial activity exhibited in the antibacterial studies. Al-ZnO nanoparticles with Cucumis maderaspatanus leaf extract produced via the green synthesis methods have remarkable antioxidant activity by scavenging free radicals against DPPH radicals, according to these results.