Aims Zinc oxide nanoparticles (ZnONPs) were successfully synthesized using the culture filtrate of the endophytic fungus Alternaria tenuissima as a rapid, eco-friendly and cost-effective method. Methods and Results The rapid synthesis of ZnONPs was completed after 20 min as confirmed by UV-Vis spectroscopy. The synthesized ZnONPs showed a single-phase crystalline structure. Dynamic light scattering analysis showed that the synthesized ZnONPs were monodispersed and the recorded polydispersity index value was 0 center dot 311. Zeta potential value of -23 center dot 92 mV indicated the high stability of ZnONPs. Transmission electron microscope revealed the spherical shape and the mean particle size was 15.45 nm. Functional groups present in the prepared samples of ZnONPs were confirmed by Fourier transform infrared spectroscopy. Additionally, the biological activities of in vitro antimicrobial, anticancer, antioxidant as well as the photocatalytic activities were evaluated. ZnONPs showed broad spectrum of antimicrobial potential against all the tested plant and human pathogens. Based on the MTT assay, ZnONPs inhibited the proliferation of normal human melanocytes, human breast and liver cancer cell lines with IC50 concentrations of 55 center dot 76, 18 center dot 02 and 16 center dot 87 mu g ml(-1). ZnONPs exhibited promising antioxidant potential with 50% inhibitory concentration of 102 center dot 13 mu g ml(-1). Moreover, ZnONPs showed efficient degradation of methylene blue dye. Conclusions The synthesized ZnONPs showed promising activities that can be better explored in the near future for many medical, agricultural and industrial applications. Significance and Impact of the Study This study suggests a new and alternate approach with the excellent biotechnological potentiality for the production of ZnONPs which could open up the way for the industrial manufacture of nanoparticles using microbial platforms.