Nanotechnology is playing a critical role in several essential technologies with nanoscale structures (nanoparticles) in areas of the environment and biomedicine. In this work, the leaf extract of Pluchea indica was utilized to biosynthesize zinc oxide nanoparticles (ZnONPs) for the first time and evaluated for antimicrobial and photocatalytic activities. Different experimental methods were used to characterize the biosynthesized ZnONPs. The biosynthesized ZnONPs showed maximum Ultraviolet–visible spectroscopy (UV-vis) absorbance at a wavelength of 360 nm. The X-Ray diffraction (XRD) pattern of the ZnONPs exhibits seven strong reflection peaks, and the average particle size was 21.9 nm. Fourier-transform infrared spectroscopy (FT-IR) spectrum analysis reveals the presence of functional groups that help in biofabrication. The existence of Zn and O was confirmed by the Energy-dispersive X-ray (EDX) spectrum and the morphology by SEM images. Antimicrobial studies showed that the biosynthesized ZnONPs have antimicrobial efficacy against Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Candida albicans and Cryptococcus neoformans where inhibition zones at concentration 1000 µg/mL were 21.83 ± 0.76, 13.0 ± 1.1, 14.9 ± 0.85, 24.26 ± 1.1, 17.0 ± 1.0, 20.67 ± 0.57 and 19.0 ± 1.0 mm respectively. Under both dark and sunlight irradiation, the photocatalytic activity of ZnONPs was evaluated towards the degradation of the thiazine dye (methylene blue-MB). Approximately 95% of the MB dye was broken down at pH 8 after 150 min of sunlight exposure. The aforementioned results, therefore, suggest that ZnONPs synthesized by implementing environmentally friendly techniques can be employed for a variety of environmental and biomedical applications.