Public health is a major concern globally, owing to the presence of industrial dyes in the effluent. Nanoparticles with green synthesis are an enthralling research field with various applications. This study deals with investigating the photocatalytic potential of Fe-oxide nanoparticles (FeO-NPs) for the degradation of methylene blue dye and their potential biomedical investigations. Biosynthesis using Anthemis tomentosa flower extract showed to be an effective method for the synthesis of FeO-NPs. The freshly prepared FeO-NPs were characterized through UV/Vis spectroscopy showing clear peak at 318 nm. The prepared FeO-NPs were of smaller size and spherical shape having large surface area and porosity with no aggregations. The FeO-NPs were characterized using XRD, FTIR, HRTEM, SEM and EDX. The HRTEM results showed that the particle size of FeO-NPs was 60-90 nm. The antimicrobial properties of FeO-NPs were investigated against two bacterial Staphylococcus aureus 13 (±0.8) and Klebsiella pneumoniae 6(±0.6) and three fungal species Aspergillus Niger, Aspergillus flavus, and Aspergillus fumigatus exhibiting a maximum reduction of 57% 47% and 50%, respectively. Moreover, FeO-NPs exhibited high antioxidant properties evaluated against ascorbic acid. Overall, this study showed high photocatalytic, antimicrobial, and antioxidant properties of FeO-NPs owing to their small size and large surface area. However, the ecotoxicity study of methylene blue degradation products showed potential toxicity to aquatic organisms.