In the era of nanotechnology, nanoparticles (NPs) of metals and metal oxides/chalcogenides are widely been used in medical applications where antibiotic-resistant microorganisms become a serious threat to the human health. Cobalt ferrite (CoFe 2 O 4) NPs, synthesized by a simple and cost-effective sol-gel auto-combustion method are envisaged for in vitro antimicrobial activities against Gram-positive bacteria (Bacillus subtilis; Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli; Pseudomonas aeruginosa). The structure, morphology, elemental analyses and surface area of CoFe 2 O 4 NPs are initially screened. The antimicrobial efficiency of CoFe 2 O 4 NPs is found to be optimum against the Gram-negative bacteria Escherichia coli (15 mm). In addition, membrane leakage assays performed to evaluate the intracellular cytoplasmic leakage with CoFe 2 O 4 NPs demonstrate the ability to destroy the bacterial membrane integrity, confirming their antimicrobial potential.