Nickel ferrite nanoparticles (NiFeO NPs) were synthesized using the medicinally important plant Aloe vera leaf extract, and their structural, morphological, and magnetic properties were characterized by x-ray diffraction (XRD), fourier transform infrared (FTIR), scanning electron microscopy (SEM), energy dispersive x-ray (EDX), and vibrating sample magnetometer (VSM). The synthesized NPs were soft ferromagnetic and spinel in nature, with an average particle size of 22.2 nm. To the best of our understanding, this is the first comprehensive investigation into the antibacterial, anticandidal, antibiofilm, and antihyphal properties of NiFeO NPs against C. albicans as well as drug-resistant gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and gram-negative multidrug resistant Pseudomonas aeruginosa (MDR-P. aeruginosa) bacteria. NiFeO NPs showed potent antimicrobial activity (MIC 1.6-2 mg/mL) against the test pathogens. NiFeO NPs at 0.5 mg/mL suppressed biofilm formation by 49.5-53.1 % in test pathogens. The study found that the NPs not only prevent the formation of biofilm, but also eliminate existing mature biofilms by 50.5-75.79 % at 0.5 mg/mL, which was further validated by SEM. SEM examination revealed a reduction in the number of cells that form biofilms and adhere to the surface. Additionally, it considerably impeded the colonization and aggregation of the biofilm strains on the glass surface. Light microscopic examination demonstrated that NPs effectively prevent the expansion of hyphae, filaments, and yeast-to-hyphae transformation in C. albicans, resulting in a substantial decrease in their ability to cause infection. Moreover, SEM images of the treated cells exhibited the presence of wrinkles, deformities, and impaired cell walls, which suggests an alteration and instability of the membrane. This study demonstrated the efficacy of the greenly manufactured NPs in suppressing the proliferation of candida, drug-resistant bacteria, and their preexisting biofilms, as well as yeast-to-hyphae transformation. Therefore, these NPs with broad spectrum applications could be utilized in health settings to mitigate biofilm-related health conditions caused by pathogenic microbial strains.