In this present work, we adopted a modified lemon juice-involved combustion method to prepare MgFe2O4 nanoparticles (MgFNPs). The prepared MgFNPs are subjected to annealing treatment at different temperatures. The synthesized and annealed MgFNPs have been studied using various techniques to analyze their structural, morphological and biomedical activities. X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the crystallite nature, shape and particles size of MgFNPs. The crystallite sizes are found in the range 10.1 nm to 32.7 nm. For various annealing temperatures, the effects of annealing treatment on structural parameters are also investigated. The spherical-shaped nanoparticle was recorded through SEM and TEM. The average size of the particles was determined from TEM micrographs using Histogram plot is 36.7 nm which is well matched with the crystallite size calculated from XRD profile. The antimicrobial activities of MgFNPs were tested against Gram-positive (B.subtilis) and Gram-negative (E.coli) bacteria by plate counting method. MgFNPs nanoparticles show strong antibacterial activity toward Gram-negative than Gram-positive bacteria. [ABSTRACT FROM AUTHOR]