Carob (Ceratonia siliqua L.) is a tree crop cultivated extensively in the eastern Mediterranean regions but that has become naturalized in other regions as well. The present study focused on the green synthesis of zinc oxide nanoparticles (ZnONPs) from Carob and their evaluation for antimicrobial activity in bacteria and fungi. The synthesized ZnONPs showed strong antibacterial activity against Staphylococcus aureus ATCC 25 923 (92%). The NPs inhibited the growth of pathogenic yeast strains, including Candida albicans ATCC90028, Candida krusei ATCC6258, and Candida neoformans ATCC14116, by 90%, 91%, and 82%, respectively, compared to the control. Fungal inhibition zones with the ZnONPs were 88.67% and 90%, respectively, larger for Aspergillus flavus 15UA005 and Aspergillus fumigatus ATCC204305, compared to control fungal growth. This study provides novel information relevant for plant-based development of new and potentially antimicrobial ZnONPs based on extracts. In particular, the development and application of phytogenic nanoparticles enhances the biocompatibility of nano-scale materials, thereby allowing to tune effects to prevent adverse outcomes in non-target biological systems.