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This investigation presents a novel finding showing the effect of culture filtrates (CFs) of macrofungi, <i>Ganoderma lucidum</i>, against <i>Meloidogyne incognita</i> evaluated in vitro and in planta. To determine the nematicidal activity, juveniles of <i>M. incognita</i> were exposed to <i>Ganoderma</i> CFs of three different ages (Two, four and eight weeks old) of pileus and stipe at different concentrations, i.e., 100%, 50%, 10% and 1% for different time intervals (12, 24, 48 and 72 h). <i>Ganoderma</i> species were examined morphologically based on external appearance and analytically using SEM. The ethanolic samples of basidiocarp were prepared and analyzed for in vitro nematicidal assay and different bioactive compounds. The in vitro experiment results revealed that among all three ages of pileus and stipe, two-week-old pileus and stipe exhibited great nematotoxic potency and caused 83.8% and 73.8% juveniles’ mortality at 100% concentration after 72 h of exposure time, respectively. Similarly, the two-week-old pileus and stipe showed the highest egg hatching inhibition of 89.2% and 81.0% at the 100% concentration after five days. The eight-week-old pileus and stipe were not more effective than the two- and four-week-old pileus and stipe. The metabolites were characterized using GC-MS, including sugar alcohol, steroids, silanes, glucosides, pyrones, ester, oleic acid, phthalic acid, linoleic acid, palmitates and ketones. The in planta study conducted in the greenhouse demonstrated that the root dip treatment for 30 min with <i>Ganoderma</i> CFs curtailed the infection level of <i>M. incognita</i> and promoted the eggplant plant growth. The maximum percent increase in plant length, plant fresh weight, plant dry weight, total chlorophyll, carotenoids and yield/plant was obtained at 100% conc. of fungus CFs, whereas a reduction was observed in nematode infestation parameters. It was concluded from the study that <i>Ganoderma</i> CFs can be explored as an effective and eco-friendly antinemic biocontrol agent in fields infected with root-knot nematodes.