Bioactive compounds and other constituents of plants have been shown to vary by cultivation region, species, environmental conditions and method of extraction among others. Phytochemical analysis of Cucurbita pepos farmed in Kiambu County, Kenya, or their seeds has not been documented. The present research aimed to bridge this knowledge gap by screening phytochemicals and characterizing the seed extracts of Cucurbita pepo cultivated in Kiambu County, Kenya. Cucurbita pepo seeds extracted using organic solvent extraction method employing methanol and preconcentrated in a vacuum rotatory evaporator. The extracts were characterized by GCMS and Fourier transform infrared (FTIR) techniques. Phytochemical analysis of the seeds revealed the presence of flavonoids, alkaloids, saponins, cardiac glycosides, and steroids. FT-IR analysis showed significant peaks for C–N, N–H, C–O, C–H, and CH3 functional groups. The GCMS studies revealed a significant number of fatty acids and their derivatives with 12-cis-octadecadienoate being the most abundant in the oil (53.93 %). A significant amount of the macrocyclic lactone 7,9-ditert-butyl-1-oxaspiro [4.5] deca-6,9-diene-2,8-dione (0.58 %) in the seeds was reported. Macrocyclic lactones are generally a class of anthelminthic drugs. These reported biologically active compounds have a wide range of medicinal and nutritional value. One interesting compound from the GCMS analysis of the seed extracts analyzed was the macrocyclic lactone providing a basis for further research on the anthelminthic actions of the seeds.