Aims Investigate the capability of Aspergillus brasiliensis ATCC 16404 to mycosynthesize Co3O4-NPs. Methods and Results Mycelial cell-free filtrate of A. brasiliensis ATCC 16404 was applied for mycosynthesis of Co3O4-NPs. The preliminary indication for the formation of Co3O4-NPs was the change in colour from yellow to reddish-brown. One-factor-at a time-optimization technique was applied to determine the optimum physicochemical conditions required for the mycosynthesis of Co3O4-NPs and they were found to be: 72 h for reaction time, pH 11, 30 degrees C, 100 rev min(-1) for shaking speed in the darkness using 4 mmol l(-1) of CoSO(4.)7H(2)O and 5 center dot 5% of A. brasiliensis dry weight mycelium (w/v). The mycosynthesized Co3O4-NPs were characterized using various techniques: spectroscopy including UV/Vis spectrophotometry, dynamic light scattering (DLS), zeta potential measurement, energy-dispersive X-ray analysis, Fourier transform infrared spectroscopy and X-ray diffraction; and vibrating sample magnetometry and microscopy including field emission scanning electron microscopy and high-resolution transmission electron microscopy. Spectroscopic techniques confirmed the formation of Co3O4-NPs and the microscopic ones confirmed the shape and size of the mycosynthesized Co3O4-NPs as quasi-spherical shaped, monodispersed nanoparticles with a nano size range of 20-27 nm. The mycosynthesized Co3O4-NPs have excellent magnetic properties and exhibited a good antimicrobial activity against some pathogenic micro-organisms. Conclusion Ferromagnetic Co3O4-NPs with considerable antimicrobial activity were for the first time mycosynthesized. Significance and Impact of the Study The use of fungi as potential bionanofactories for mycosynthesis of nanoparticles is relatively a recent field of research with considerable prospects.